Mammalian cytokines; related reagents and methods

ABSTRACT

Purified genes encoding cytokine from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding this molecule are provided. Methods of using said reagents and diagnostic kits are also provided.

[0001] This filing is a conversion to U.S. Utility Patent Application ofU.S.SNo. 60/124,319, filed Mar. 11, 1999, which is incorporated hereinby reference.

FIELD OF THE INVENTION

[0002] The present invention pertains to compositions related toproteins which function in controlling biology and physiology ofmammalian cells, e.g., cells of a mammalian immune system. Inparticular, it provides purified genes, proteins, antibodies, relatedreagents, and methods useful, e.g., to regulate activation, development,differentiation, and function of various cell types, includinghematopoietic cells.

BACKGROUND OF THE INVENTION

[0003] Recombinant DNA technology refers generally to the technique ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

[0004] For some time, it has been known that the mammalian immuneresponse is based on a series of complex cellular interactions, calledthe “immune network”. See, e.g., Paul (1998) Fundamental Immunology (4thed.) Raven Press, NY. Recent research has provided new insights into theinner workings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which will lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system disorders. Some of thesefactors are hematopoietic growth factors, e.g., granulocyte colonystimulating factor (G-CSF). See, e.g., Thomson (ed. 1998) The CytokineHandbook (3d ed.) Academic Press, San Diego; Mire-Sluis and Thorpe (ed.1998) Cytokines Academic Press, San Diego; Metcalf and Nicola (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press, andAggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

[0005] Lymphokines apparently mediate cellular activities in a varietyof ways. They have been shown to support the proliferation, growth,and/or differentiation of pluripotential hematopoietic stem cells intovast numbers of progenitors comprising diverse cellular lineages makingup a complex immune system. Proper and balanced interactions between thecellular components are necessary for a healthy immune response. Thedifferent cellular lineages often respond in a different manner whenlymphokines are administered in conjunction with other agents.

[0006] Cell lineages especially important to the immune response includetwo classes of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

[0007] Research to better understand and treat various immune disordershas been hampered by the general inability to maintain cells of theimmune system in vitro. Immunologists have discovered that culturingthese cells can be accomplished through the use of T-cell and other cellsupernatants, which contain various growth factors, including many ofthe lymphokines.

[0008] From the foregoing, it is evident that the discovery anddevelopment of new lymphokines, e.g., related to G-CSF and/or IL-6,could contribute to new therapies for a wide range of degenerative orabnormal conditions which directly or indirectly involve the immunesystem and/or hematopoietic cells. In particular, the discovery anddevelopment of lymphokines which enhance or potentiate the beneficialactivities of known lymphokines would be highly advantageous. Thepresent invention provides new interleukin compositions and relatedcompounds, and methods for their use.

SUMMARY OF THE INVENTION

[0009] The present invention is directed to mammalian, e.g., primate orrodent, interleukin-B60 (IL-B60) and its biological activities. Itincludes nucleic acids coding for polypeptides themselves and methodsfor their production and use. The nucleic acids of the invention arecharacterized, in part, by their homology to complementary DNA (cDNA)sequences disclosed herein, and/or by functional assays for growthfactor- or cytokine-like activities, e.g., G-CSF (see Nagata (1994) inThomson The Cytokine Handbook 2d ed., Academic Press, San Diego) and/orIL-6 (see Hirano (1994) in Thomson The Cytokine Handbook 2d ed.,Academic Press, San Diego). Also provided are polypeptides, antibodies,and methods of using them, including using nucleic acid expressionmethods. Methods for modulating or intervening in the control of agrowth factor dependent physiology or an immune response are provided.

[0010] The present invention is based, in part, upon the discovery of anew cytokine sequence exhibiting significant sequence and structuralsimilarity to G-CSF and IL-6. In particular, it provides primate, e.g.,human, and rodent, e.g., mouse, genes encoding a protein whose maturesize is about 198 amino acids. Functional equivalents exhibitingsignificant sequence homology will be available from other mammalian,e.g., cow, horse, and rat species.

[0011] Moreover, the present invention identifies a second associatedcomponent of a complex. Compositions related to the combination ofcomponents in the complex are provided, along with methods of use.

[0012] In one embodiment, the invention provides a substantially pure orrecombinant polypeptide comprising the mature protein portion of SEQ IDNO: 2 or 4. Preferably, the polypeptide is: detectably labeled;unglycosylated; denatured; attached to a solid substrate; conjugated toanother chemical moiety; or in a sterile composition. Kit forms includethose comprising the polypeptide and: a compartment comprising thepolypeptide; or with instructions for use or disposal of reagents in thekit.

[0013] Binding compounds include those comprising an antigen bindingsite from an antibody that specifically binds to the describedpolypeptide. The binding compound can also be in a kit comprising:a-compartment comprising the binding compound; or with instructions foruse or disposal of reagents in the kit.

[0014] The invention further provides a method of producing anantigen:antibody complex, comprising contacting, under appropriateconditions, a primate IL-B60 polypeptide with an antibody thatspecifically or selectively binds the polypeptide of the invention,thereby allowing the complex to form.

[0015] Nucleic acid embodiments include an isolated or recombinantpolynucleotide encoding the mature protein portion of SEQ ID NO: 2 or 4.

[0016] In other embodiments, the invention provides an isolated solublecomplex comprising the mature protein portion of SEQ ID NO: 2 or 4, andthe mature protein portion of SEQ ID NO: 12 or 13. Preferably thecomplex: comprises a recombinant polypeptide of SEQ ID NO: 2, 4, 12, or13; is detectably labeled; is in a buffered solution; is in a sterilesolution. Kits are provided containing such a complex and: a compartmentcomprising the complex; or instructions for use or disposal of reagentsin the kit.

[0017] Binding compounds are provided comprising an antigen binding sitefrom an antibody that specifically binds to the soluble complex but notto the mature polypeptide of SEQ ID NO: 12 or 13. Kits are providedcomprising the binding compound and: a compartment comprising thebinding compound; or instructions for use or disposal of reagents in thekit.

[0018] Methods are provided, e.g., of producing an antigen:antibodycomplex, comprising contacting, under appropriate conditions, a primatecomplex comprising IL-B60 and CLF-1 polypeptides with an antibody thatselectively or specifically binds to an isolated soluble complexcomprising the mature protein portion of SEQ ID NO: 2 or 4, and themature protein portion of SEQ ID NO: 12 or 13, thereby allowing thecomplex to form.

[0019] Nucleic acid embodiments include an isolated or recombinantnucleic acid encoding the mature protein portion of SEQ ID NO: 2 or 4,and the mature protein portion of SEQ ID NO: 12 or 13.

[0020] The invention also provides a composition of matter selectedfrom: an isolated polypeptide comprising at least seven amino acidsidentical to segments of SEQ ID NO: 2 or 4; a substantially pure orrecombinant polypeptide comprising at least two distinct nonoverlappingsegments of at least five amino acids identical to segments of SEQ IDNO: 2 or 4; a natural sequence polypeptide comprising mature SEQ ID NO:2 or 4; or a fusion polypeptide comprising IL-B60 sequence. In certainembodiments, the distinct nonoverlapping segments of identity include:one of at least eight amino acids; one of at least five amino acids anda second of at least six amino acids; at least three segments of atleast four, five, and six amino acids, or one of at least twelve aminoacids. In other embodiments the polypeptide of the composition ofmatter: is the polypeptide which: comprises a mature sequence of Table1; is an unglycosylated form of IL-B60; is from a primate, such as ahuman; comprises at least seventeen amino acids of SEQ ID NO: 2 or 4;exhibits at least four nonoverlapping segments of at least seven aminoacids of SEQ ID NO: 2 or 4; is a natural allelic variant of IL-B60; hasa length at least about 30 amino acids; exhibits at least twonon-overlapping epitopes which are specific for a primate IL-B60; isglycosylated; has a molecular weight of at least 30 kD with naturalglycosylation; is a synthetic polypeptide; is attached to a solidsubstrate; is conjugated to another chemical moiety; is a 5-fold or lesssubstitution from natural sequence; is a deletion or insertion variantfrom a natural sequence; or which further comprises: at least sevenamino acids identical to segments of SEQ ID NO: 12 or 13; at least twodistinct nonoverlapping segments of at least five amino acids identicalto segments of SEQ ID NO: 12 or 13; a natural sequence polypeptidecomprising mature SEQ ID NO: 12 or 13; or a primate CLF-1. In additionalpreferred embodiments, the composition comprises: a substantially pureIL-B60 and CLF-1; a sterile IL-B60 polypeptide comprising the matureprotein of SEQ ID NO: 2 or 4; or the described polypeptide and acarrier, wherein the carrier is: an aqueous compound, including water,saline, and/or buffer; and/or formulated for oral, rectal, nasal,topical,mRNA,or parenteral administration. The invention provides fusionpolypeptides which comprise: mature protein sequence of Table 1; adetection or purification tag, including a FLAG, His6, or Ig sequence;or sequence of another cytokine receptor family protein, includingCLF-1. Kit embodiments include those comprising the polypeptide of thecomposition and: a compartment comprising the protein or polypeptide; orinstructions for use or disposal of reagents in the kit.

[0021] The invention further provides methods of using the describedpolypeptides: to label the polypeptide, comprising labeling thepolypeptide with a radioactive label; to separate the polypeptide fromanother polypeptide in a mixture, comprising running the mixture on achromatography matrix, thereby separating the polypeptides; to identifya compound that binds selectively to the polypeptide, comprisingincubating the compound with the polypeptide under appropriateconditions; thereby causing the component to bind to the polypeptide; orto conjugate the polypeptide to a matrix, comprising derivatizing thepolypeptide with a reactive reagent, and conjugating the polypeptide tothe matrix..

[0022] Related binding compounds include those comprising an antigenbinding site from an antibody that specifically or selectively binds toa natural polypeptide, as described above, wherein: the binding compoundis in a container; the IL-B60 polypeptide is from a human; the bindingcompound is an Fv, Fab, or Fab2 fragment; the binding compound isconjugated to another chemical moiety; or the antibody: is raisedagainst a mature polypeptide of Table 1; is raised against a matureIL-B60; is raised to a purified human IL-B60; is immunoselected; is apolyclonal antibody; binds to a denatured IL-B60; exhibits a Kd toantigen of at least 30 μM; is attached to a solid substrate, including abead or plastic membrane; is in a sterile composition; or is detectablylabeled, including a radioactive or fluorescent label. Kits are providedcomprising such a binding compound and: a compartment comprising thebinding compound; or instructions for use or disposal of the reagents ofthe kit.

[0023] Methods are provided for producing an antigen:antibody complex,comprising contacting, under appropriate conditions, a primate IL-B60polypeptide with a described antibody, thereby allowing the complex toform. Preferably, in the method: the complex is purified from othercytokines; the complex is purified from other antibody; the contactingis with a sample comprising a cytokine; the contacting allowsquantitative detection of the antigen; the contacting is with a samplecomprising the antibody; or the contacting allows quantitative detectionof the antibody.

[0024] In another embodiment the invention includes a compositioncomprising: a sterile binding compound, as described, or the bindingcompound and a carrier, wherein the carrier: wherein the carrier is: anaqueous compound, including water, saline, and/or buffer; and/orformulated for oral, rectal, nasal, topical, or parenteraladministration.

[0025] Nucleic acid embodiments include an isolated or recombinantnucleic acid encoding the described polypeptide, wherein: the IL-B60 isfrom a human; or the nucleic acid: encodes an antigenic peptide sequenceof Table 1; encodes a plurality of antigenic peptide sequences of Table1; encodes a plurality of antigenic peptide sequences of Table 4;exhibits identity over at least thirteen nucleotides to a natural cDNAencoding the segment; is an expression vector; further comprises anorigin of replication; is from a natural source; comprises a detectablelabel; comprises synthetic nucleotide sequence; is less than 6 kb,preferably less than 3 kb; is from a primate; comprises a natural fulllength coding sequence; is a hybridization probe for a gene encoding theIL-B60; or is a PCR primer, PCR product, or mutagenesis primer.Preferred embodiments include where the isolated or recombinant nucleicacid is in a cell or tissue. The cell may be: a prokaryotic cell; aeukaryotic cell; a bacterial cell; a yeast cell; an insect cell; amammalian cell; a mouse cell; a primate cell; or a human cell.

[0026] Kits are provided comprising the described nucleic acid and: acompartment comprising the nucleic acid; a compartment furthercomprising a primate IL-B60 polypeptide; or instructions for use ordisposal of reagents in the kit.

[0027] The invention further provides methods for forming a duplex witha polynucleotide described above, comprising contacting thepolynucleotide with a probe that hybridizes, under stringent conditions,to at least 25 contiguous nucleotides of the coding portion of SEQ IDNO: 1, 3, or encoding the nature SEQ ID NO: 12 or 13; thereby formingthe duplex.

[0028] In a further aspect, the invention provides a nucleic acid which:hybridizes under wash conditions of 30 minutes at 30° C. and less than2M salt to the coding portion of SEQ ID NO: 1; or exhibits identityover-a stretch of at least about 30 nucleotides to a primate IL-B60. Inpreferred embodiments, the wash conditions that are at 45° C. and/or 500mM salt; or at 55° C. and/or 150 mM salt; or the stretch is at least 55nucleotides, e.g., at least 75 nucleotides.

[0029] Methods are provided, e.g., of modulating physiology ordevelopment of a cell or tissue culture cells comprising contacting thecell with an agonist or antagonist of a mammalian IL-B60; or contactingthe cell with an agonist or antagonist of a complex comprising mammalianIL-B60 and CLF-1. Additionally, the invention provides a method ofincreasing the secretion of: an IL-B60 sequence, comprising expressingthe polypeptide with CLF-1; or a CLF-1, comprising expressing the CLF-1with an IL-B60 sequence. In preferred embodiments of the method, theincreasing is at least 3 fold, 5×, 7×, 10×, or more; or the expressingis of a recombinant nucleic acid encoding one or both of the polypeptideand CLF-1.

[0030] The invention further provides a method of screening for areceptor which binds an isolated soluble complex comprising the matureprotein portion of SEQ ID NO: 2 or 4, and the mature protein portion ofSEQ ID NO: 12 or 13, comprising contacting the complex to a cellexpressing the receptor under conditions allowing the complex to bind tothe receptor, thereby forming a detectable interaction. Preferably, theinteraction results in a physiological response in the cell.

[0031] Other embodiments of the invention include, e.g., an isolatedsoluble complex comprising at least 6 amino acids of the mature proteinportion of SEQ ID NO: 2 or 4, and: at least 6 amino acids of the matureprotein portion of SEQ ID NO: 12 or 13; or at least 6 amino acids of themature protein portion of the CNTF-R. Such complex may, e.g., comprise arecombinant polypeptide of mature SEQ ID NO: 2 or 4; comprise arecombinant polypeptide of mature SEQ ID NO: 12 or 13; comprise arecombinant polypeptide of mature CNTF-R; comprise both a recombinantpolypeptide of mature SEQ ID NO: 2 or 4, and a recombinant polypeptideof mature SEQ ID NO: 12 or 13; comprise both a recombinant polypeptideof mature SEQ ID NO: 2 or 4, and a recombinant polypeptide of matureCNTF-R; be detectably labeled; be in a buffered solution; or be in asterile solution. Preferred embodiments include those which: comprise amature IL-B60 polypeptide; comprise a mature CLF-1 polypeptide;comprises a mature CNTF-R polypeptide; exhibit at least fournonoverlapping segments of at least seven amino acids of SEQ ID NO: 2 or4; exhibit epitopes from both primate L-B60 and primate CLF-1; exhibitepitopes from both primate L-B60 and primate CNTF-R; not beglycosylated; be attached to a solid substrate; be conjugated to anotherchemical moiety; or comprise a detection or purification tag, includinga FLAG, His6, or Ig sequence.

[0032] Kits are provided, e.g., comprising the complex and: acompartment comprising the complex, and/or instructions for use ordisposal of reagents in the kit.

[0033] Fusion polypeptides are provided, which include, e.g., anisolated or recombinant polypeptide comprising: a first segmentcomprising at least seven amino acids identical to segments of SEQ IDNO: 2 or 4, and a second segment comprising at least seven amino acidsidentical to segments of mature SEQ ID NO: 12 or 13; at least twodistinct nonoverlapping segments of at least five amino acids identicalto segments of mature SEQ ID NO: 2 or 4, and a third segment comprisingat least seven amino acids identical to segments of mature SEQ ID NO: 12or 13; at least one segment comprising at least seven amino acidsidentical to segments of mature SEQ ID NO: 2 or 4, and two distinctnonoverlapping segments of at least five amino acids identical tosegments of mature SEQ ID NO: 12 or 13; a first segment comprising atleast seven amino acids identical to segments of SEQ ID NO: 2 or 4, anda second segment comprising at least seven amino acids identical tosegments of mature primate CNTF-R; at least two distinct nonoverlappingsegments of at least five amino acids identical to segments of matureSEQ ID NO: 2 or 4, and a third segment comprising at least seven aminoacids identical to segments of mature primate CNTF-R; or at least onesegment comprising at least seven amino acids identical to segments ofmature SEQ ID NO: 2 or 4, and two distinct nonoverlapping segments of atleast five amino acids identical to segments of mature primate CNTF-R.Certain embodiments include those wherein the distinct nonoverlappingsegments of identity: include one of at least eight amino acids; includeone of at least five amino acids and a second of at least six aminoacids; include at least three segments of at least four, five, and sixamino acids, or include one of at least twelve amino acids. Otherembodiments include those which: comprise a mature IL-B60 sequence;comprise a mature CLF-1 sequence; comprise a mature CNTF-R sequence;exhibit at least four nonoverlapping segments of at least seven aminoacids of SEQ ID NO: 2 or 4; have a length at least about 30 amino acids;exhibit epitopes from both primate IL-B60 and primate CLF-1; exhibitsepitopes from both primate IL-B60 and primate CNTF-R; are notglycosylated; have a molecular weight of at least 30 kD; be a syntheticpolypeptide; be attached to a solid substrate; be conjugated to anotherchemical moiety; or comprise a detection or purification tag, includinga FLAG, His6, or Ig sequence.

[0034] Other embodiments include a composition comprising: substantiallypure combination of IL-B60 and CLF-l; substantially pure combination ofIL-B60 and CNTF-R; a sterile polypeptide described above; or thepolypeptide described above and a carrier, wherein the carrier is: anaqueous compound, including water, saline, and/or buffer; and/orformulated for oral, rectal, nasal, topical, or parenteraladministration. A kit is provided comprising a polypeptide as described,and: a compartment comprising the polypeptide; and/or instructions foruse or disposal of reagents in the kit.

[0035] Methods are also provided, e.g., of making an antibody whichrecognizes a complex as described, comprising inducing an immuneresponse in an animal with the complex; of immunoselecting antibodies,comprising contacting a population of antibodies to a complex asdescribed, and separating antibodies that bind from those which do notbind; or of formulating a composition, comprising admixing a complex asdescribed with a carrier.

[0036] Binding compounds are provided, e.g., comprising an antigenbinding site from an antibody, which antibody specifically binds adescribed complex, but not to any of the mature polypeptides of SEQ IDNO: 2, 4, 12, 13, or CNTF-R. Certain embodiments include those wherein:the binding compound is: in a container; an Fv, Fab, or Fab2 fragment;or conjugated to another chemical moiety; or the antibody: is raisedagainst a substantially pure complex of IL-B60 with CLF-1; is raisedagainst a substantially pure complex of IL-B60 with CNTF-R; isimmunoselected; is a polyclonal antibody; exhibits a Kd to antigen of atleast 30 μM; is attached to a solid substrate, including a bead orplastic membrane; is in a sterile composition; or is detectably labeled,including a radioactive or fluorescent label. Additional embodimentsinclude a composition comprising: a sterile binding compound asdescribed, or the binding compound as described and a carrier, whereinthe carrier is: an aqueous compound, including water, saline, and/orbuffer; and/or formulated for oral, rectal, nasal, topical, orparenteral administration.

[0037] With the binding composition are provided a kit comprising thebinding compound and: a compartment comprising the binding compound; orinstructions for use or disposal of reagents in the kit. Also providedare methods of producing an antigen:antibody complex, comprisingcontacting under appropriate conditions a primate complex comprising:IL-B60 and CLF-1 polypeptides; or IL-B60 and CNTF-R polypeptides; withan antibody as described, thereby allowing the complex to form.Preferably, in the method, the complex is purified from other cytokines;the complex is purified from other antibody; the contacting is with asample comprising a cytokine; the contacting allows quantitativedetection of the antigen; the contacting is with a sample comprising theantibody; or the contacting allows quantitative detection of theantibody.

[0038] Various nucleic acids are provided, e.g., an isolated orrecombinant nucleic acid: encoding the amino acid portions describedabove; encoding the amino acid portions as described, and comprise asegment at least 30 contiguous nucleotides from SEQ ID NO: 1 or 3; whichwill coexpress a segment of at least seven contiguous amino acids fromSEQ ID NO: 2 or 4, and a segment of at least seven contiguous aminoacids from SEQ ID NO: 12 or 13; or which will coexpress a segment of atleast seven contiguous amino acids from SEQ ID NO: 2 or 4, and a segmentof at least seven contiguous amino acids from CNTF-R. Preferred nucleicacids include those which, e.g.,: encode IL-B60 from a human; encodeCLF-1 from a human; encodes CNTF-R from a human; are an expressionvector; further comprise an origin of replication; comprise a detectablelabel; comprise synthetic nucleotide sequence; or are less than 6 kb,preferably less than 3 kb. A cell comprising the recombinant nucleicacid is provided, e.g., wherein the cell is: a prokaryotic cell; aeukaryotic cell; a bacterial cell; a yeast cell; an insect cell; amammalian cell; a mouse cell; a primate cell; or a human cell. Variousnucleic acid kits are provided, e.g., comprising the nucleic acid and: acompartment comprising the nucleic acid; a compartment furthercomprising a primate IL-B60 polypeptide; a compartment furthercomprising a primate CLF-1 polypeptide; a compartment further comprisinga primate CNTF-R polypeptide; or instructions for use or disposal ofreagents in the kit. Methods are also provided, e.g., of making a duplexnucleic acid, comprising contacting such a nucleic acid with acomplementary nucleic acid under appropriate conditions, thereby formingsaid duplex; of expressing a polypeptide, comprising expressing thenucleic acid, thereby producing the polypeptide; or of transfecting acell, comprising contacting said cell under appropriate conditions withthe nucleic acid, thereby transfecting the cell.

[0039] In an alternative embodiment, the invention provides an isolatedor recombinant nucleic acid which encodes at least 5 contiguous aminoacids of SEQ ID NO: 12, 13, or primate CNTF-R and: hybridizes under washconditions of 30 minutes at 30° C. and less than 2M salt to the codingportion of SEQ ID NO: 1; or exhibits identity over a stretch of at leastabout 30 nucleotides to a primate IL-B60. Preferred embodiments include:the isolated nucleic acid, wherein: the contiguous amino acids number atleast 8; the wash conditions are at 45° C. and/or 500 mM salt; or thestretch is at least 55 nucleotides; or the recombinant nucleic acid,wherein: the contiguous amino acids number at least 12; the washconditions are at 55° C. and/or 150 mM salt; or the stretch is at least75 nucleotides.

[0040] The invention particularly provides methods of modulatingphysiology or development of a cell or tissue culture cells comprisingcontacting the cell with an agonist or antagonist of a complexcomprising mammalian IL-B60 and: CLF-1; or CNTF-R. It also providesmethods of producing the proteins, e.g., producing a complex described,comprising coexpressing a recombinant IL-B60 with a recombinant CLF-1 orCNTF-R; increasing the secretion of an IL-B60 polypeptide comprisingexpressing the polypeptide with CLF-1 or CNTF-R; or increasing thesecretion of a CLF-1 polypeptide, comprising expressing the CLF-1 withan IL-B60. Typically, the increasing is at least 3 fold; or theexpressing is of a recombinant nucleic acid encoding one or both of thepolypeptide and CLF-1.

[0041] Also provided are methods of screening for a receptor which bindsthe described complex, comprising contacting the complex to a cellexpressing the receptor under conditions allowing the complex to bind tothe receptor, thereby forming a detectable interaction. Preferably, theinteraction results in a physiological response in the cell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] All references cited herein are incorporated herein by referenceto the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

[0043] Outline I. General II. Purified IL-B60 or complex A. physicalproperties B. biological properties III. Physical Variants A. sequencevariants, fragments B. post-translational variants 1.  glycosylation2.  others IV. Functional Variants A. analogs, fragments 1.  agonists2.  antagonists B. mimetics 1.  protein 2.  chemicals C. speciesvariants V. Antibodies A. polyclonal B. monoclonal C. fragments, bindingcompositions VI. Nucleic Acids A. natural isolates; methods B. syntheticgenes C. methods to isolate VII. Making IL-B60 or complex, mimetics A.recombinant methods B. synthetic methods C. natural purification VIII.Uses A. diagnostic B. therapeutic IX. Kits A. nucleic acid reagents B.protein reagents C. antibody reagents X. Isolating receptors for IL-B60or complex

[0044] I. General

[0045] The present invention provides amino acid sequences and DNAsequences encoding various mammalian proteins which are cytokines, e.g.,which are secreted molecules which can mediate a signal between immuneor other cells. See, e.g., Paul (1998) Fundamental Immunology (4th ed.)Raven Press, N.Y. The full length cytokines, and fragments, orantagonists will be useful, e.g., in physiological modulation of cellsexpressing a receptor. It is likely that IL-B60 has either stimulatoryor inhibitory effects on hematopoietic cells, including, e.g., lymphoidcells, such as T-cells, B-cells, natural killer (NK) cells, macrophages,dendritic cells, hematopoietic progenitors, etc. The proteins will alsobe useful as antigens, e.g., immunogens, for raising antibodies tovarious epitopes on the protein, both linear and conformationalepitopes.

[0046] A sequence encoding IL-B60 was identified from a human genomicsequence. The molecule was designated huIL-B60. A rodent sequence, e.g.,from mouse, is also described.

[0047] The human gene encodes a small soluble cytokine-like protein, ofabout 198 amino acids. The psort predicted signal sequence probably isabout 17 residues, and would run from the Met to about Ala. See Table 1and SEQ. ID. NO: 1 and 2. IL-B60 exhibits structural motifscharacteristic of a member of the long chain cytokines. Compare, e.g.,IL-B60, G-CSF, and IL-6, sequences available from GenBank. Closestmatching is with CT-1, oncostatin M, and CNTF. See also Table 2. TABLE 1Nucleic acid (SEQ ID NO: 1) encoding IL-B60 from a primate, e.g., human.Predicted signal cleavage site is indicated. Nucleotide 375 may be A.Translated amino acid sequence is SEQ ID NO: 2. ccgagcgaaa aaaacctgcgagtgggcctg gcggatggga ttattaaagc ttcgccggag   60 ccgcggctcg ccctcccactccgccagcct ccgggagagg agccgcaccc ggccggcccg  120 gccccagccc catggacctccgagcagggg actcgtgggg g atg tta gcg tgc ctg  176                                              Met Leu Ala Cys Leu                                                      −15 tgc acg gtgctc tgg cac ctc cct gca gtg cca gct ctc aat cgc aca  224 Cys Thr Val LeuTrp His Leu Pro Ala Val Pro Ala Leu Asn Arg Thr        −10                  −5              −1   1 ggg gac cca ggg cctggc ccc tcc atc cag aaa acc tat gac ctc acc  272 Gly Asp Pro Gly Pro GlyPro Ser Ile Gln Lys Thr Tyr Asp Leu Thr  5                  10                  15                  20 cgc tacctg gag cac caa ctc cgc agc ttg gct ggg acc tat ctg aac  320 Arg Tyr LeuGlu His Gln Leu Arg Ser Leu Ala Gly Thr Tyr Leu Asn                 25                  30                  35 tac ctg ggcccc cct ttc aac gag cca gac ttc aac cct ccc cgc ctg  368 Tyr Leu Gly ProPro Phe Asn Glu Pro Asp Phe Asn Pro Pro Arg Leu             40                  45                  50 ggg gca gag actctg ccc agg gcc act gtt gac ttg gag gtg tgg cga  416 Gly Ala Glu Thr LeuPro Arg Ala Thr Val Asp Leu Glu Val Trp Arg         55                  60                  65 agc ctc aat gac aaactg cgg ctg acc cag aac tac gag gcc tac agc  464 Ser Leu Asn Asp Lys LeuArg Leu Thr Gln Asn Tyr Glu Ala Tyr Ser     70                  75                  80 cac ctt ctg tgt tac ttgcgt ggc ctc aac cgt cag gct gcc act gct  512 His Leu Leu Cys Tyr Leu ArgGly Leu Asn Arg Gln Ala Ala Thr Ala 85                  90                  95                 100 gag ctgcgc cgc agc ctg gcc cac ttc tgc acc agc ctc cag ggc ctg  560 Glu Leu ArgArg Ser Leu Ala His Phe Cys Thr Ser Leu Gln Gly Leu                105                 110                 115 ctg ggc agcatt gcg ggc gtc atg gca gct ctg ggc tac cca ctg ccc  608 Leu Gly Ser IleAla Gly Val Met Ala Ala Leu Gly Tyr Pro Leu Pro            120                 125                 130 cag ccg ctg cctggg act gaa ccc act tgg act cct ggc cct gcc cac  656 Gln Pro Leu Pro GlyThr Glu Pro Thr Trp Thr Pro Gly Pro Ala His        135                 140                 145 agt gac ttc ctc cagaag atg gac gac ttc tgg ctg ctg aag gag ctg  704 Ser Asp Phe Leu Gln LysMet Asp Asp Phe Trp Leu Leu Lys Glu Leu    150                 155                 160 cag acc tgg ctg tgg cgctcg gcc aag gac ttc aac cgg ctc aag aag  752 Gln Thr Trp Leu Trp Arg SerAla Lys Asp Phe Asn Arg Leu Lys Lys165                 170                 175                 180 aag atgcag cct cca gca gct gca gtc acc ctg cac ctg ggg gct cat  800 Lys Met GlnPro Pro Ala Ala Ala Val Thr Leu His Leu Gly Ala His                185                 190                 195 ggc ttctgacttctga ccttctcctc ttcgctcccc cttcaaaccc tgctcccact  856 Gly Phettgtgagagc cagccctgta tgccaacacc tgttgagcca ggagacagaa gctgtgagcc  916tctggccctt tcctggaccg gctgggcgtg tgatgcgatc agccctgtct cctccccacc  976tcccaaaggt ctaccgagct ggggaggagg tacagtaggc cctgtcctgt cctgtttcta 1036caggaagtca tgctcgaggg agtgtgaagt ggttcaggtt ggtgcagagg cgctcatggc 1096ctcctgcttc ttgcctacca cttggccagt gcccacccag cccctcaggt ggcacatctg 1156gagggcaggg gttgaggggc caccaccaca catgcctttc tggggtgaag ccctttggct 1216gccccactct ccttggatgg gtgttgctcc cttatcccca aatcactcta tacatccaat 1276tcaggaaaca aacatggtgg caattctaca caaaaagaga tgagattaac agtgcagggt 1336tggggtctgc attggaggtg ccctataaac cagaagagaa aatactgaaa gcacaggggc 1396agggacagac cagaccagac ccaggagtct ccaaagcaca gagtggcaaa caaaacccga 1456gctgagcatc aggaccttgc ctcgaattgt cttccagtat tacggtgcct cttctctgcc 1516ccctttccca gggtatctgt gggttgccag gctggggagg gcaaccatag ccacaccaca 1576ggatttcctg aaagtttaca atgcagtagc attttggggt gtagggtggc agctccccaa 1636ggccctgccc cccagcccca cccactcatg actctaagtg tgttgtatta atatttattt 1696atttggagat gttatttatt agatgatatt tattgcagaa tttctattct tgtattaaca 1756aataaaatgc ttgccccaga acaaaaaaaa aaaa 1790MLACLCTVLWHLPAVPA/LNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVDLEVWRSLNDKLRLTQNYEAYSHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSIAGVMAALGYPLPQPLPGTEPTWTPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAAAVTLHLGAHGF Rodent, e.g., mouse, IL-B60 (SEQ ID NO: 3 and 4): atg tta gcttgc cta tgc acg gtg ctg tgg cac ctc cct gca gtg cca   48 Met Leu Ala CysLeu Cys Thr Val Leu Trp His Leu Pro Ala Val Pro        −15                 −10                  −5 gct ctt aat cgc acagga gat cca ggc cct ggc ccc tcc atc cag aaa   96 Ala Leu Asn Arg Thr GlyAsp Pro Gly Pro Gly Pro Ser Ile Gln Lys -1   1               5                  10                  15 acc tatgac ctc acc cgc tac ctg gag cat caa ctc cgc agc tta gct  144 Thr Tyr AspLeu Thr Arg Tyr Leu Glu His Gln Leu Arg Ser Leu Ala                 20                  25                  30 ggg acc tacctg aac tac ctg ggg ccc cct ttc aac gag cct gac ttc  192 Gly Thr Tyr LeuAsn Tyr Leu Gly Pro Pro Phe Asn Glu Pro Asp Phe             35                  40                  45 aat cct cct cgactg ggg gca gaa act ctg ccc agg gcc acg gtc aac  240 Asn Pro Pro Arg LeuGly Ala Glu Thr Leu Pro Arg Ala Thr Val Asn         50                  55                  60 ttg gaa gtg tgg cgaagc ctc aat gac agg ctg cgg ctg acc cag aac  288 Leu Glu Val Trp Arg SerLeu Asn Asp Arg Leu Arg Leu Thr Gln Asn     65                  70                  75 tat gag gcg tac agt cacctc ctg tgt tac ttg cgt ggc ctc aac cgt  336 Tyr Glu Ala Tyr Ser His LeuLeu Cys Tyr Leu Arg Gly Leu Asn Arg 80                  85                  90                  95 cag gctgcc aca gct gaa ctc cga cgt agc ctg gcc cac ttc tgt acc  384 Gln Ala AlaThr Ala Glu Leu Arg Arg Ser Leu Ala His Phe Cys Thr                100                 105                 110 agc ctc cagggc ctg ctg ggc agc att gca ggt gtc atg gcg acg ctt  432 Ser Leu Gln GlyLeu Leu Gly Ser Ile Ala Gly Val Met Ala Thr Leu            115                 120                 125 ggc tac cca ctgccc cag cct ctg cca ggg act gag cca gcc tgg gcc  480 Gly Tyr Pro Leu ProGln Pro Leu Pro Gly Thr Glu Pro Ala Trp Ala        130                 135                 140 cct ggc cct gcc cacagt gac ttc ctc cag aag atg gat gac ttc tgg  528 Pro Gly Pro Ala His SerAsp Phe Leu Gln Lys Met Asp Asp Phe Trp    145                 150                 155 ctg ctg aag gag ctg cagacc tgg cta tgg cgt tca gcc aag gac ttc  576 Leu Leu Lys Glu Leu Gln ThrTrp Leu Trp Arg Ser Ala Lys Asp Phe160                 165                 170                 175 aac cggctt aag aag aag atg cag cct cca gca gct tca gtc acc ctg  624 Asn Arg LeuLys Lys Lys Met Gln Pro Pro Ala Ala Ser Val Thr Leu                180                 185                 190 cac ttg gaggcc cat ggt ttc tga  648 His Leu Glu Ala His Gly Phe             195MLACLCTVLWHLPAVPA/LNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVNLEVWRSLNDRLRLTQNYEAYSHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSIAGVMATLGYPLPQPLPGTEPAWAPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAASVTLHLEAHGF

[0048] TABLE 2 Comparison of primate and rodent embodiments of IL-B60,both the nucleotide and amino acid sequences. hIL-B60ATGTTAGCGTGCCTGTGCACGGTGCTCTGGCACCTCCCTGCAGTGCCAGCTCTCAATCGC mIL-B60ATGTTAGCTTGCCTATGCACGGTGCTGTGGCACCTCCCTGCAGTGCCAGCTCTTAATCGC ************* *********** ************************** ****** hIL-B60ACAGGGGACCCAGGGCCTGGCCCCTCCATCCAGAAAACCTATGACCTCACCCGCTACCTG mIL-B60ACAGGAGATCCAGGCCCTGGCCCCTCCATCCAGAAAACCTATGACCTCACCCGCTACCTG ***** ******* ********************************************* hIL-B60GAGCACCAACTCCGCAGCTTGGCTGGGACCTATCTGAACTACCTGGGCCCCCCTTTCAAC mIL-B60GAGCATCAACTCCGCAGCTTAGCTGGGACCTACCTGAACTACCTGGGGCCCCCTTTCAAC ******************* *********** ************** ************ hIL-B60GAGCCAGACTTCAACCCTCCCCGCCTGGGGGCAGAGACTCTGCCCAGGGCCACTGTTGAC mIL-B60GAGCCTGACTTCAATCCTCCTCGACTGGGGGCAGAAACTCTGCCCAGGGCCACGGTCAAC ************* ***** ** *********** ***************** **  ** hIL-B60TTGGAGGTGTGGCGAAGCCTCAATGACAAACTGCGGCTGACCCAGAACTACGAGGCCTAC mIL-B60TTGGAAGTGTGGCGAAGCCTCAATGACAGGCTGCGGCTGACCCAGAACTATGAGGCGTAC ***************************  ******************** ***** *** hIL-B60AGCCACCTTCTGTGTTACTTGCGTGGCCTCAACCGTCAGGCTGCCACTGCTGAGCTGCGC mIL-B60AGTCACCTCCTGTGTTACTTGCGTGGCCTCAACCGTCAGGCTGCCACAGCTGAACTCCGA ** ******************************************* ***** ** ** hIL-B60CGCAGCCTGGCCCACTTCTGCACCAGCCTCCAGGGCCTGCTGGGCAGCATTGCGGGCGTC mIL-B60CGTAGCCTGGCCCACTTCTGTACCAGCCTCCAGGGCCTGCTGGGCAGCATTGCAGGTGTC ******************* ******************************** ** *** hIL-B60ATGGCAGCTCTGGGCTACCCACTGCCCCAGCCGCTGCCTGGGACTGAACCCACTTGGACT mIL-B60ATGGCGACGCTTGGCTACCCACTGCCCCAGCCTCTGCCAGGGACTGAGCCAGCCTGGGCC *****  * ********************** ***** ******** **  * *** * hIL-B60CCTGGCCCTGCCCACAGTGACTTCCTCCAGAAGATGGACGACTTCTGGCTGCTGAAGGAG mIL-B60CCTGGCCCTGCCCACAGTGACTTCCTCCAGAAGATGGATGACTTCTGGCTGCTGAAGGAG************************************** ********************* hIL-B60CTGCAGACCTGGCTGTGGCGCTCGGCCAAGGACTTCAACCGGCTCAAGAAGAAGATGCAG mIL-B60CTGCAGACCTGGCTATGGCGTTCAGCCAAGGACTTCAACCGGCTTAAGAAGAAGATGCAG************** ***** ** ******************** *************** hIL-B60CCTCCAGCAGCTGCAGTCACCCTGCACCTGGGGGCTCATGGCTTCTGA mIL-B60CCTCCAGCAGCTTCAGTCACCCTGCACTTGGAGGCCCATGGTTTCTGA ************************** *** *** ***** ****** Alignment of IL-B60: underlined areproposed helices. In general, those residues that are in helix A and Dand not pointing inward toward the core (mostly the hydrophobic residuesin A and D helix) are the most likely residues to interact withreceptors.                                       A hIL-B60MLACLCTVLWHLPAVPALNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGT mIL-B60MLACLCTVLWHLPAVPALNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGT**************************************************                                            B hIL-B60YLNYLGPPFNEPDFNPPRLGAETLPRATVDLEVWRSLNDKLRLTQNYEAY mIL-B60YLNYLGPPFNEPDFNPPRLGAETLPRATVNLEVWRSLNDRLRLTQNYEAY*****************************:*********:**********                                      C hIL-B60SHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSIAGVMAALGYPLPQ mIL-B60SHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSIAGVMATLGYPLPQ******************************************:*******                                      D hIL-B60PLPGTEPTWTPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQ mIL-B60PLPGTEPAWAPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQ*******:*:**************************************** hIL-B60PPAAAVTLHLGAHGF mIL-B60 PPAASVTLHLEAHGF ****:***** ****

[0049] TABLE 3 Comparison of various cytokines compared to IL-B60. HumanIL-B60 is SEQ ID NO: 2; mouse IL-B60 is SEQ ID NO: 4; mouse LIF (mLIF)is SEQ ID NO: 5 and Accession number X06381; human LIF (hLIF) is SEQ IDNO: 6 and Accession numbers M63420 J05436; human CT-1 (hCT-1) is SEQ IDNO: 7 and Accession number U43030; mouse CT-1 (mCT-1) is SEQ ID NO: 8and Accession number U18366; human CNTF (hCNTF) is SEQ ID NO: 9 andAccession number X60542; mouse CNTF (mCNTF) is SEQ ID NO: 10 andAccession number U05342; human DNAX IL-40 (hDIL-40) is SEQ ID NO: 11.mLIF -MKVLAAGIVPLLLLVLHWKHGAGSPLPI-TPVNATC-AIRHPCHGNLMN hLIF-MKVLAAGVVP-LLLVLHWKHGAGSPLPI-TPVNATC-AIRHPCHNNLMN hCT-1--MSRREGSLE---D--PQTDSSVSLLPH-LEA-----KIRQT-HS--LA mCT-1--MSQREGSLE---D--HQTDSSISFLPH-LEA-----KIRQT-HN--LA hIL-B60-MLACLCTVLW------HLPAVPALNRTG-DPG-PGP-SIQKT-YD--LT mIL-B60-MLACLCTVLW------HLPAVPALNRTG-DPG-PGP-SIQKT-YD--LT hCNTF------MAFTE------HSPLTPHR-R---D-L-CSR-SIW-------LA mCNTF------MAFAE------QSPLTLHR-R---D-L-CSR-SIW-------LA hDIL-40MTHLSLLGPLPCVRTSQQLPETQQVTTPGKKPVSVGRREVRVP-----GT                                       :     . mLIFQIKNQLAQLNQSANALFISYYTAQGEPF--PNNVEK-LCAPNMTDFPSFH hLIFQIRSQLAQLNGSANALFILYYTAQGEPF--PNNLDK-LCGPNVTDFPPFH hCT-1HLLTKYAEQ------LLQEYVQLQGDPFGLPSFSPPRLPVAGLSAPAPSH mCT-1RLLTKYAEQ------LLEEYVQQQGEPFGLPGFSPPRLPLAGLSGPAPSH hIL-B60RYLEHQLRS------LAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVDL mIL-B60RYLEHQLRS------LAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVNL hCNTFRKIRSDLTA------LTESYVKHQG--LNK---NINLDSADGMPVASTD- mCNTFRKIRSDLTA------LMESYVKHQG--LNK---NISLDSVDGVPVASTD- hDIL-40ALVPSLLSV------SVLLQLQYQGSPFSDPGFSAPELQLSSLPPATAFF                        *  :.  .          :.  . mLIF---GNGTEKTKLVELYRMVAYLSASLTNITR-DQKVLNPTAVSLQVKLNA hLIF---ANGTEKAKLVELYRIVVYLGTSLGNITR-DQKILNPSALSLHSKLNA hCT-1---AGLPVHERLRLDAAALAALPPLLDAVCR-RQAELNPRAPRLLRRLED mCT-1---AGLPVSERLRQDAAALSVLPALLDAVRR-RQAELNPRAPRLLRSLED hIL-B60EVWRSLNDKLRLTQNYEAYSHLLCYLRGLN--RQAATAELRRSLAHFCTS mIL-B60EVWRSLNDRLRLTQNYEAYSHLLCYLRGLN--RQAATAELRRSLAHFCTS hCNTF-QWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHT mCNTF-RWSEMTEAERLQENLQAYRTFQGMLTKLLEDQRVHFTPTEGDFHQAIHT hDIL-40KTWHALDDGERLSLAQRAID---PHLQLVED-DQSDLNPGSPILPAQLGA:         :*             *  :    :         : mLIFTIDVMRGLLSNVLCRLCNKYRV--GHVDVPP-----VPDHSDKE--AFQR hLIFTADILRGLLSNVLCRLCSKYHV--GHVDVTY-----GPDTSGKD--VFQK hCT-1AARQARALGAAVEALLAALGAANRGPRAEPP--AATASAASATG--VFPA mCT-1AARQVRALGAAVETVLAALGAAARGPGPEPVTVATLFTANSTAG--IFSA hIL-B60LQGLLGSIAGVMAALGYPLPQP--LPGTEPT----WTPGPAHS---DFLQ mIL-B60LQGLLGSIAGVMATLGYPLPQP--LPGTEPA----WAPGPAHS---DFLQ hCNTFLLLQVAAFAYQIEELMILLEYK--IPRNEAD----GMPINVGDGG-LFEK mCNTFLTLQVSAFAYQLEELMALLEQK--VPEKEAD----GMPVTIGDGG-LFEK hDIL-40ARLRAQGPLGNMAAIMTALGLP--IP-PEED-----TPGLAAFGASAFER      .    :                         .         * mLIFKKLGCQLLGTYKQVIS----VVVQAF--------------------- hLIFKKLGCQLLGKYKQIIA----VLAQAF--------------------- hCT-1KVLGLRVCGLYREWLSRTEGDLGQLLPGGSA---------------- mCT-1KVLGFHVCGLYGEWVSRTEGDLGQLVPGGVA---------------- hIL-B60KMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAAAVTLHLGAHGF-- mIL-B60KMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAASVTLHLEAHGF-- hCNTFKLWGLKVLQELSQWTVRSIHDL-RFISSHQTGIPARGSHYIANNKKM mCNTFKLWGLKVLQELSQWTVRSIHDL-RVISSHHMGISAHESHYGA--KQM hDIL-40KCRGYVVTREYGHWTDRAVRDLALLKAKYSA----------------*  .  :        .     .                :   :.

[0050] The structural homology of IL-B60 to related cytokine proteinssuggests related function of this molecule. IL-B60 is a long chaincytokine exhibiting sequence similarity to IL-6 and G-CSF.

[0051] IL-B60 agonists, or antagonists, may also act as functional orreceptor antagonists, e.g., which block IL-6 or G-CSF binding to theirrespective receptors, or mediating the opposite actions. Thus, IL-B60,or its antagonists, may be useful in the treatment of abnormal medicalconditions, including immune disorders, e.g., T cell immunedeficiencies, chronic inflammation, or tissue rejection, or incardiovascular or neurophysiological conditions.

[0052] The natural antigens are capable of mediating various biochemicalresponses which lead to biological or physiological responses in targetcells. The preferred embodiment characterized herein is from human, butother primate, or other species counterparts exist in nature. Additionalsequences for proteins in other mammalian species, e.g., primates,canines, felines, and rodents, should also be available. See below. Thedescriptions below are directed, for exemplary purposes, to a humanIL-B60, but are likewise applicable to related embodiments from otherspecies.

[0053] In particular, the association of the IL-B60 with a partner hasbeen confirmed. The IL-B60 and CLF-1 molecules have likely evolvedtogether, reflected in their homology between species. For example, thecoevolution of their function is suggested by the observation that thehuman/mouse relationship of the IL-B60 is very close, as is thehuman/mouse CLF-1. If the two functionally associate, they might acttogether in the fashion of IL-12. See, e.g., Trinchieri (1998) Adv.Immunol. 70:83-243; Gately, et al.(1998) Ann. Rev. Immunol. 16:495-521;and Trinchieri (1998) Int. Rev. Immunol. 16:365-396.

[0054] As a complex, however, they will interact with two tall receptorsin the cytokine receptor family, e.g., gp130, LIF-R, OSM-R, IL-12Rb1,IL-12Rb2, and NR30. These receptors will be tested for binding to thesoluble complex. A series of BAF/3 cells that stably express various ofthese tall receptors have been constructed.

[0055] The supernatants of transfectants of both IL-B60 and CLF-1 (or asingle combination construct) in the same cell, will be used to testthese various BAF/3 cells to see if there is a proliferative or othersignaling response. As such, most of the physiological effects of thecytokine may be due to the complex of the proteins. As such, many of thedescriptions below of biology resulting from the cytokine may actuallybe physiologically effected by the complex comprising the combination ofthe subunits.

[0056] Table 4 provides the sequences of the IL-B60 partner, known asCLF-1. The CNTF receptor (CNTF-R) subunit alpha was described, e.g., byDavis, et al. (1991) Science 253:59-63. See also GenBank accessionnumbers NM1001842 and M73238 (human); AF068615 (mouse); and S54212(rat); each of which is incorporated herein by reference. TABLE 4Alignment of human and mouse Cytokine-Like Factor 1 (CLF-1; SEQ ID NO:12 and 13) See Elson, et al. (1998) J. Immunol. 161:1371-1379; GenBankAccession number AF059293 and NM_004750; also described by Douglas J.Hilton (Australia) in WO9920755. Reported signal sequence of 37 aminoacids in human form, cleavage at GSG/AHT. hCLF-1MPAGRRGPAAQSARRPPPLLPLLLLLCVLGAPRAGSGAHTAVISPQDPTL mCLF-1--------------RPLSSLWSPLLLCVLGVPRGGSGAHTAVISPQDPTL               ** .*   *******.**.**************** hCLF-1LIGSSLLATCSVHGDPPGATAEGLYWTLNGRRLPPELSRVLNASTLALAL mCLF-1LIGSSLQATCSIHGDTPGATAEGLYWTLNGRRLP-SLSRLLNTSTLALAL **********:***.****************** .***:**:******* hCLF-1ANLNGSRQRSGDNLVCHARDGSILAGSCLYVGLPPEKPVNISCWSKNMKD mCLF-1ANLNGSRQQSGDNLVCHARDGSILAGSCLYVGLPPEKPFNISCWSRNMKD********:*****************************.******.**** hCLF-1LTCRWTPGAHGETFLHTNYSLKYKLRWYGQDNTCEEYHTVGPHSCHIPKD mCLF-1LTCRWTPGAHGETFLHTNYSLKYKLRWYGQDNTCEEYHTVGPHSCHIPKD************************************************** hCLF-1LALFTPYEIWVEATNRLGSARSDVLTLDILDVVTTDPPPDVHVSRVGGLE mCLF-1LALFTPYEIWVEATNRLGSARSDVLTLDVLDVVTTDPPPDVHVSRVGGLE****************************:********************* hCLF-1DQLSVRWVSPPALKDFLFQAKYQIRYRVEDSVDWKVVDDVSNQTSCRLAG mCLF-1DQLSVRWVSPPALKDFLFQAKYQIRYRVEDSVDWKVVDDVSNQTSCRLAG************************************************** hCLF-1LKPGTVYFVQVRCNPFGIYGSKKAGIWSEWSHPTAASTPRSERPGPGGGA mCLF-1LKPGTVYFVQVRCNPFGIYGSKKAGIWSEWSHPTAASTPRSERPGPGGGV*************************************************. hCLF-1CEPRGGEPSSGPVRRELKQFLGWLKKHAYCSNLSFRLYDQWRAWMQKSHK mCLF-1CEPRGGEPSSGPVRRELKQFLGWLKKHAYCSNLSFRLYDQWRAWMQKSHK************************************************** hCLF-1TRNQ---VLPDKL--------- mCLF-1 TRNQDEGILPSGRRGAARGPAG ****   :**.

[0057] Standard domains of the human CLF-1 receptor sequence correspondapproximately to: signal from 1 to about 38; first IG-like domain fromabout residue 39 to 130; a second domain from about 131. to about 237;and the last from about 238 to the end.

[0058] The descriptions below may also be applied to the CLF-1, or tothe IL-B60/CLF-1 complex. A fusion of the IL-B60 with CLF-1 may beconstructed, as, e.g., the hyper IL-6. See, e.g., Fischer, et al. (1997)Nature Biotechnol. 15:142-145; Rakemann, et al. (1999) J. Biol. Chem.274:1257-1266; and Peters, et al.(1998) J. Immunol. 161:3575-3581; whichare incorporated herein by reference.

[0059] The original discovery and molecular characterization of CNTF asa potent survival factor for neuronal cells (see, e.g., Hughes, et al.(1988) Nature 335:70-73; and Stockli, et al. (1989) Nature 342:920-923)suggested a prospective therapeutic use as a molecule that could speedrepair of damaged or severed motor neurons (Sendtner, et al. (1990)Nature 345:440-441; and Curtis, et al. (1993) Nature 365:253-256) orprevent nerve degeneration (Sendtner, et al. (1992) Nature 358:502-504;Emerich, et al. (1997) Nature 386:395-399; and Gravel, et al. (1997)Nature Med. 3:765-770). However, CNTF is oddly a protein without asecretory signal peptide, and does not appear to escape the cell(Stockli, et al., ibid); furthermore, engineered (Masu, et al. (1993)Nature 365:27-32) or naturally occurring (Takahashi, et al. (1994)Nature Genet. 7:79-84) disruptions of the CNTF gene are not deleterious.By contrast, gene disruptions of the primary receptor for CNTF (CNTF-Rα)prove lethal shortly after birth. DeChiara, et al. (1995) Cell83:313-322). Together, these observations point to the existence of asecond ligand for CNTF-Rα that is physiologically responsible for the invitro observed, or in vivo desired, actions of CNTF. This workdemonstrates that the composite cytokine IL-B60/CLF-1 is likely thislong sought-after factor that is both developmentally critical, it issecreted from target organs and directs their innervation by motorneurons, as well as therapeutically promising, since nerve transectionresults in a fast and. long lasting induction of both IL-B60 and CLF-1,indicating a role for the complex in regeneration. In support of thismodel, gene disruption of CLF-1 (Alexander, et al.. (1999) Curr. Biol.9:605-608) is quite similar in phenotype to the CNTF-Rα knock-out.

[0060] In an intriguing twist, while IL-B60 has a signal peptide, itssecretion remains critically dependent on complexing with CLF-1, asdescribed. Once secreted, IL-B60 signals via a tripartite receptorsystem that is otherwise identical to that of CNTF, consisting of thetwo ubiquitously expressed signal-transducing components gp130 andLIF-R, and the specificity-determining receptor, CNTF-Rα. Strikingly,the role of CLF-1 seems to be restricted to that of a chaperone, sinceit is discarded from the signaling complex after delivering IL-B60 toCNTF-Rα; indeed, the requirement for CLF-1 can be sidestepped by fusingIL-B60 directly to a soluble form of the CNTF-Rα chain. All threeprotein chains involved in this novel system, IL-B60, CLF-1, andCNTF-Rα, represent the most highly conserved sequences in thehematopoietic cytokine/receptor superfamily, indicating anevolutionarily critical interaction. Furthermore, the conditional use ofa hematopoietic receptor as a secretion factor and escort presents anovel paradigm for cytokine activity.

[0061] To summarize, this work sheds light on the entwined biologicalfunction of two orphan molecules, IL-B60 and CLF-1, by describing theirnovel engagement of the CNTF receptor complex. In doing so, we present astrong argument that it is IL-B60/CLF-1 cytokine that serves as a keyphysiological factor in motor neuron development and regeneration.

[0062] II. Purified IL-B60 or Complex

[0063] Human IL-B60 amino acid sequence, is shown, in one embodimentwithin SEQ ID NO: 2. Other naturally occurring nucleic acids whichencode the protein can be isolated by standard procedures using theprovided sequence, e.g., PCR techniques, or by hybridization. Theseamino acid sequences, provided amino to carboxy, are important inproviding sequence information for the cytokine allowing fordistinguishing the protein antigen from other proteins and exemplifyingnumerous variants. Moreover, the peptide sequences allow preparation ofpeptides to generate antibodies to recognize such segments, andnucleotide sequences allow preparation of oligonucleotide probes, bothof which are strategies for detection or isolation, e.g., cloning, ofgenes encoding such sequences.

[0064] As used herein, the term “human soluble IL-B60” shall encompass,when used in a protein context, a protein having amino acid sequencecorresponding to a soluble polypeptide from SEQ ID NO: 2. Significantfragments thereof will often retain similar functions, e.g.,antigenicity. Preferred embodiments comprise a plurality of distinct,e.g., nonoverlapping, segments of the specified length. Typically, theplurality will be at least two, more usually at least three, andpreferably 5, 7, or even more. While the length minima may be recited,longer lengths, of various sizes, may be appropriate, e.g., one oflength 7, and two of length 12. Similar features apply topolynucleotides.

[0065] Binding components, e.g., antibodies, typically bind to an IL-B60with high affinity, e.g., at least about 100 nM, usually better thanabout 30 nM, preferably better than about 10 nM, and more preferably atbetter than about 3 nM. Counterpart proteins will be found in mammalianspecies other than human, e.g., other primates, ungulates, or rodents.Non-mammalian species should also possess structurally or functionallyrelated genes and proteins, e.g., birds or amphibians.

[0066] The term “polypeptide” as used herein includes a significantfragment or segment, and encompasses a stretch of amino acid residues ofat least about 8 amino acids, generally at least about 12 amino acids,typically at least about 16 amino acids, preferably at least about 20amino acids, and, in particularly preferred embodiments, at least about30 or more amino acids, e.g., 35, 40, 45, 50, etc. Such fragments mayhave ends which begin and/or end at virtually all positions, e.g.,beginning at residues 1, 2, 3, etc., and ending at, e.g., 150, 149, 148,etc., in all practical combinations. Particularly interesting peptideshave ends corresponding to structural domain boundaries, e.g., helicesA, B, C, and/or D. See Table 1.

[0067] The term “binding composition” refers to molecules that bind withspecificity to IL-B60, e.g., in an antibody-antigen interaction. Thespecificity may be more or less inclusive, e.g., specific to aparticular embodiment, or to groups of related embodiments, e.g.,primate, rodent, etc. Depletion or absorptions can provide desiredselectivities. Also provided are compounds, e.g., proteins, whichspecifically associate with IL-B60, including in a naturalphysiologically relevant protein-protein interaction, either covalent ornon-covalent. The molecule may be a polymer, or chemical reagent. Afunctional analog may be a protein with structural modifications, or itmay be a molecule which has a molecular shape which interacts with theappropriate binding determinants. The compounds may serve as agonists orantagonists of a receptor binding interaction, see, e.g., Goodman, etal. (eds.) Goodman & Gilman's: The Pharmacological Bases of Therapeutics(current ed.) Pergamon Press.

[0068] Substantially pure, e.g., in a protein context, typically meansthat the protein is free from other contaminating proteins, nucleicacids, or other biologicals derived from the original source organism.Purity may be assayed by standard methods, typically by weight, and willordinarily be at least about 40% pure, generally at least about 50%pure, often at least about 60% pure, typically at least about 80% pure,preferably at least about 90% pure, and in most preferred embodiments,at least about 95% pure. Carriers or excipients will often be added.

[0069] Solubility of a polypeptide or fragment depends upon theenvironment and the polypeptide. Many parameters affect polypeptidesolubility, including temperature, electrolyte environment, size andmolecular characteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

[0070] The size and structure of the polypeptide should generally be ina substantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents.

[0071] The solvent and electrolytes will usually be a biologicallycompatible buffer, of a type used for preservation of biologicalactivities, and will usually approximate a physiological aqueoussolvent. Usually the solvent will have a neutral pH, typically betweenabout 5 and 10, and preferably about 7.5. On some occasions, one or moredetergents will be added, typically a mild non-denaturing one, e.g., CHS(cholesteryl hemisuccinate) or CHAPS(3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), or a lowenough concentration as to avoid significant disruption of structural orphysiological properties of the protein. In other instances, a harshdetergent may be used to effect significant denaturation.

[0072] An IL-B60 polypeptide that specifically binds to or that isspecifically immunoreactive with an antibody, e.g., such as a polyclonalantibody, generated against a defined immunogen, e.g., such as animmunogen consisting of an amino acid sequence of SEQ ID NO: 2 orfragments thereof or a polypeptide generated from the nucleic acid ofSEQ ID NO: 1 is typically determined in an immunoassay. Included withinthe metes and bounds of the present invention are those nucleic acidsequences described herein, including functional variants, that encodepolypeptides that selectively bind to polyclonal antibodies generatedagainst the prototypical IL-B60 polypeptide as structurally andfunctionally defined herein. The immunoassay typically uses a polyclonalantiserum which was raised, e.g., to a protein of SEQ ID NO: 2. Thisantiserum is selected, or depleted, to have low crossreactivity againstappropriate other closely related family members, preferably from thesame species, and any such crossreactivity is removed byimmunoabsorption or depletion prior to use in the immunoassay.Appropriate selective serum preparations can be isolated, andcharacterized.

[0073] In order to produce antisera for use in an immunoassay, theprotein, e.g., of SEQ ID NO: 2, is isolated as described herein. Forexample, recombinant protein may be produced in a mammalian cell line.An appropriate host, e.g., an inbred strain of mice such as Balb/c, isimmunized with the protein of SEQ ID NO: 2 using a standard adjuvant,such as Freund's adjuvant, and a standard mouse immunization protocol(see Harlow and Lane). Alternatively, a substantially full lengthsynthetic peptide derived from the sequences disclosed herein can beused as an immunogen. Polyclonal sera are collected and titered againstthe immunogen protein in an immunoassay, e.g., a solid phase immunoassaywith the immunogen immobilized on a solid support. Polyclonal antiserawith a titer of 10⁴ or greater are selected and tested for their crossreactivity against other closely related family members, e.g., LIF,CT-1, CNTF, DIL-40, or other members of the IL-6 family, using acompetitive binding immunoassay such as the one described in Harlow andLane, supra, at pages 570-573. Preferably at least two IL-6/IL-12 familymembers are used in this determination in conjunction with the target.These long chain cytokine family members can be produced as recombinantproteins and isolated using standard molecular biology and proteinchemistry techniques as described herein. Thus, antibody preparationscan be identified or produced having desired selectivity or specificityfor subsets of IL-B60 family members. Alternatively, antibodies may beprepared which bind to the complex comprising the IL-B60 with the CLF-1.

[0074] Immunoassays in the competitive binding format can be used forthe crossreactivity determinations. For example, the protein of SEQ IDNO: 2 can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the protein of SEQ IDNO: 2. The percent crossreactivity for the above proteins is calculated,using standard calculations. Those antisera with less than 10%crossreactivity with each of the proteins listed above are selected andpooled. The cross-reacting antibodies are then removed from the pooledantisera by immunoabsorption with the above-listed proteins.

[0075] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein to the immunogen protein. In order to make this comparison, thetwo proteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required is less than twice the amount of the protein of,e.g., SEQ ID NO: 2 that is required, then the second protein is said tospecifically bind to an antibody generated to the immunogen.

[0076] III. Physical Variants

[0077] This invention also encompasses proteins or peptides havingsubstantial amino acid sequence identity with the amino acid sequence ofthe IL-B60 antigen. The variants include species, polymorphic, orallelic variants.

[0078] Amino acid sequence homology, or sequence identity, is determinedby optimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. The conservation may apply tobiological features, functional features, or structural features.Homologous amino acid sequences are typically intended to includenatural polymorphic or allelic and interspecies variations of a proteinsequence. Typical homologous proteins or peptides will have from 25-100%identity (if gaps can be introduced), to 50-100% identity (ifconservative substitutions are included) with the amino acid sequence ofthe IL-B60. Identity measures will be at least about 35%, generally atleast about 40%, often at least about 50%, typically at least about 60%,usually at least about 70%, preferably at least about 80%, and morepreferably at least about 90%.

[0079] The isolated IL-B60 DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of short nucleotide stretches. These modifications result innovel DNA sequences which encode these antigens, their derivatives, orproteins having similar physiological, immunogenic, antigenic, or otherfunctional activity. These modified sequences can be used to producemutant antigens or to enhance expression. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. “Mutant IL-B60” encompasses apolypeptide otherwise falling within the sequence identity definition ofthe IL-B60 as set forth above, but having an amino acid sequence whichdiffers from that of IL-B60 as normally found in nature, whether by wayof deletion, substitution, or insertion. This generally includesproteins having significant identity with a protein having sequence ofSEQ ID NO: 2, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most of the natural full length disclosed sequences.Full length sequences will typically be preferred, though truncatedversions will also be useful, likewise, genes or proteins found fromnatural sources are typically most desired. Similar concepts apply todifferent IL-B60 proteins, particularly those found in various warmblooded animals, e.g., mammals and birds. These descriptions aregenerally meant to encompass all IL-B60 proteins, not limited to theparticular primate embodiments specifically discussed.

[0080] IL-B60 mutagenesis can also be conducted by making amino acidinsertions or deletions. Substitutions, deletions, insertions, or anycombinations may be generated to arrive at a final construct. Insertionsinclude amino- or carboxy-terminal fusions. Random mutagenesis can beconducted at a target codon and the expressed mutants can then bescreened for the desired activity. Methods for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown in the art, e.g., by M13 primer mutagenesis or polymerase chainreaction (PCR) techniques. See, e.g., Sambrook, et al. (1989); Ausubel,et al. (1987 and Supplements); and Kunkel, et al. (1987) Methods inEnzymol. 154:367-382. Preferred embodiments include, e.g., 1-fold,2-fold, 3-fold, 5-fold, 7-fold, etc., preferably conservativesubstitutions at the nucleotide or amino acid levels. Preferably thesubstitutions will be away from the conserved cysteines, and often willbe in the regions away from the helical structural domains. Suchvariants may be useful to produce specific antibodies, and often willshare many or all biological properties. See Table 2. Recognition of thecytokine structure provides important insight into the structure andpositions of residues which may be modified to effect desired changes inreceptor interaction. Also, the interaction of the IL-B60 with the CLF-1protein requires complementary structural features in the interactingsurface.

[0081] The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences.

[0082] In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.

[0083] The phosphoramidite method described by Beaucage and Carruthers(1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

[0084] Structural analysis can be applied to this gene, in comparison tothe IL-6 family of cytokines. The family includes, e.g., IL-6, IL-11,IL-12, G-CSF, LIF, OSM, CNTF, and Ob. Alignment of the human and mouseIL-B60 sequences with other members of the IL-6 family should allowdefinition of structural features. In particular, β-sheet and α-helixresidues can be determined using, e.g., RASMOL program, see Bazan, etal. (1996) Nature 379:591; Lodi, et al. (1994) Science 263:1762-1766;Sayle and Milner-White (1995) TIBS 20:374-376; and Gronenberg, et al.(1991) Protein Engineering 4:263-269. See, also, Wilkins, et al. (eds.1997) Proteome Research: New Frontiers in Functional GenomicsSpringer-Verlag, NY. Preferred residues for substitutions include thesurface exposed residues which would be predicted to interact withreceptor. Other residues which should conserve function will beconservative substitutions, particularly at position far from thesurface exposed residues.

[0085] IV. Functional Variants

[0086] The blocking of physiological response to IL-B60s may result fromthe competitive inhibition of binding of the ligand to its receptor.

[0087] In vitro assays of the present invention will often use isolatedprotein, soluble fragments comprising receptor binding segments of theseproteins, or fragments attached to solid phase substrates. These assayswill also allow for the diagnostic determination of the effects ofeither binding segment mutations and modifications, or cytokinemutations and modifications, e.g., IL-B60 analogs.

[0088] This invention also contemplates the use of competitive drugscreening assays, e.g., where neutralizing antibodies to the cytokine,or receptor binding fragments compete with a test compound.

[0089] “Derivatives” of IL-B60 antigens include amino acid sequencemutants from naturally occurring forms, glycosylation variants, andcovalent or aggregate conjugates with other chemical moieties. Covalentderivatives can be prepared by linkage of functionalities to groupswhich are found in IL-B60 amino acid side chains or at the N- orC-termini, e.g., by standard means. See, e.g., Lundblad and Noyes (1988)Chemical Reagents for Protein Modification, vols. 1-2, CRC Press, Inc.,Boca Raton, Fla.; Hugli (ed. 1989) Techniques in Protein Chemistry,Academic Press, San Diego, Calif.; and Wong (1991) Chemistry of ProteinConjugation and Cross Linking, CRC Press, Boca Raton, Fla.

[0090] In particular, glycosylation alterations are included, e.g., madeby modifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0091] Fusion polypeptides between IL-B60s and other homologous orheterologous proteins are also provided. Many cytokine receptors orother surface proteins are multimeric, e.g., homodimeric entities, and arepeat construct may have various advantages, including lessenedsusceptibility to proteolytic cleavage. Typical examples are fusions ofa reporter polypeptide, e.g., luciferase, with a segment or domain of aprotein, e.g., a receptor-binding segment, so that the presence orlocation of the fused ligand may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609. Other gene fusion partners includebacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase,alcohol dehydrogenase, yeast alpha mating factor, and detection orpurification tags such as a FLAG sequence of His6 sequence. See, e.g.,Godowski, et al. (1988) Science 241:812-816.

[0092] Fusion peptides will typically be made by either recombinantnucleic acid methods or by synthetic polypeptide methods. Techniques fornucleic acid manipulation and expression are described generally, e.g.,in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2ded.), vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al.(eds. 1993) Current Protocols in Molecular Biology, Greene and Wiley,NY. Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341-347; Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W. H. Freeman, NY. Refolding methodsmay be applicable to synthetic proteins.

[0093] This invention also contemplates the use of derivatives of IL-B60proteins other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties or protein carriers. Covalent or aggregativederivatives will be useful as immunogens, as reagents in immunoassays,or in purification methods such as for affinity purification of bindingpartners, e.g., other antigens. An IL-B60 can be immobilized by covalentbonding to a solid support such as cyanogen bromide-activated SEPHAROSE,by methods which are well known in the art, or adsorbed onto polyolefinsurfaces, with or without glutaraldehyde cross-linking, for use in theassay or purification of anti-IL-B60 antibodies or an alternativebinding composition. The IL-B60 proteins can also be labeled with adetectable group, e.g., for use in diagnostic assays. Purification ofIL-B60 may be effected by an immobilized antibody or complementarybinding partner, e.g., binding portion of a receptor.

[0094] A solubilized IL-B60 or fragment of this invention can be used asan immunogen for the production of antisera or antibodies specific forbinding. Purified antigen can be used to screen monoclonal antibodies orantigen-binding fragments, encompassing antigen binding fragments ofnatural antibodies, e.g., Fab, Fab′, F(ab)2, etc. Purified IL-B60antigens can also be used as a reagent to detect antibodies generated inresponse to the presence of elevated levels of the cytokine, which maybe diagnostic of an abnormal or specific physiological or diseasecondition. This invention contemplates antibodies raised against aminoacid sequences encoded by nucleotide sequence shown in SEQ ID NO: 1, orfragments of proteins containing it. In particular, this inventioncontemplates antibodies having binding affinity to or being raisedagainst specific domains, e.g., helices A, B, C, or D of the IL-B60, orthe Ig domains of the CLF-1.

[0095] The present invention contemplates the isolation of additionalclosely related species variants. Southern and Northern blot analysiswill establish that similar genetic entities exist in other mammals. Itis likely that IL-B60s are widespread in species variants, e.g.,rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, andprimates.

[0096] The invention also provides means to isolate a group of relatedantigens displaying both distinctness and similarities in structure,expression, and function. Elucidation of many of the physiologicaleffects of the molecules will be greatly accelerated by the isolationand characterization of additional distinct species or polymorphicvariants of them. In particular, the present invention provides usefulprobes for identifying additional homologous genetic entities indifferent species.

[0097] The isolated genes will allow transformation of cells lackingexpression of an IL-B60, e.g., either species types or cells whichlack-corresponding proteins and exhibit negative background activity.This should allow analysis of the function of IL-B60 in comparison tountransformed control cells.

[0098] Dissection of critical structural elements which effect thevarious physiological functions mediated through these antigens ispossible using standard techniques of modern molecular biology,particularly in comparing members of the related class. See, e.g., thehomolog-scanning mutagenesis technique described in Cunningham, et al.(1989) Science 243:1339-1336; and approaches used in O'Dowd, et al.(1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et al. (1990)EMBO J. 9:4381-4390.

[0099] Intracellular functions would probably involve receptorsignaling. However, protein internalization may occur under certaincircumstances, and interaction between intracellular components andcytokine may occur. Specific segments of interaction of IL-B60 withinteracting components may be identified by mutagenesis or directbiochemical means, e.g., cross-linking or affinity methods. Structuralanalysis by crystallographic or other physical methods will also beapplicable. Further investigation of the mechanism of signaltransduction will include study of associated components which may beisolatable by affinity methods or by genetic means, e.g.,complementation analysis of mutants.

[0100] Further study of the expression and control of IL-B60 will bepursued. The controlling elements associated with the antigens shouldexhibit differential physiological, developmental, tissue specific, orother expression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest.

[0101] Structural studies of the IL-B60 antigens will lead to design ofnew antigens, particularly analogs exhibiting agonist or antagonistproperties on the molecule. This can be combined with previouslydescribed screening methods to isolate antigens exhibiting desiredspectra of activities.

[0102] V. Antibodies

[0103] Antibodies can be raised to various epitopes of the mRNA* IL-B60proteins, including species, polymorphic, or allelic variants, andfragments thereof, both in their naturally occurring forms and in theirrecombinant forms. Additionally, antibodies can be raised to IL-B60s ineither their active forms or in their inactive forms, including nativeor denatured versions. Anti-idiotypic antibodies are also contemplated.

[0104] Antibodies, including binding fragments and single chainversions, against predetermined fragments of the antigens can be raisedby immunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective IL-B60s, or screened for agonistic orantagonistic activity, e.g., mediated through a receptor. Antibodies maybe agonistic or antagonistic, e.g., by sterically blocking binding to areceptor. These monoclonal antibodies will usually bind with at least aK_(D) of about 1 mM, more usually at least about 300 μM, typically atleast about 100 μM, more typically at least about 30 μM, preferably atleast about 10 μM, and more preferably at least about 3 μM or better.

[0105] The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingto a receptor. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying IL-B60 protein or its receptors. See, e.g., Chan (ed. 1987)Immunology: A Practical Guide, Academic Press, Orlando, Fla.; Price andNewman (eds. 1991) Principles and Practice of Immunoassay, StocktonPress, N.Y.; and Ngo (ed. 1988) Nonisotopic Immunoassay, Plenum Press,N.Y. Cross absorptions or other tests will identify antibodies whichexhibit various spectra of specificities, e.g., unique or shared speciesspecificities.

[0106] Further, the antibodies, including antigen binding fragments, ofthis invention can be potent antagonists that bind to the antigen andinhibit functional binding, e.g., to a receptor which may elicit abiological response. They also can be useful as non-neutralizingantibodies and can be coupled to toxins or radionuclides so that whenthe antibody binds to antigen, a cell expressing it, e.g., on itssurface, is killed. Further, these antibodies can be conjugated to drugsor other therapeutic agents, either directly or indirectly by means of alinker, and may effect drug targeting.

[0107] Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams, et al.(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane (1988) Antibodies:. A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

[0108] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basicand Clinical Immunology (4th ed.), Lange Medical Publications, LosAltos, Calif., and references cited therein; Harlow and Lane (1988)Antibodies: A Laboratory Manual, CSH Press; Goding (1986) MonoclonalAntibodies: Principles and Practice (2d ed.), Academic Press, New York;and particularly in Kohler and Milstein (1975) in Nature 256:495-497,which discusses one method of generating monoclonal antibodies.

[0109] Other suitable techniques involve in vitro exposure oflymphocytes to the antigenic polypeptides or alternatively to selectionof libraries of antibodies in phage or similar vectors. See, Huse, etal. (1989) “Generation of a Large Combinatorial Library of theImmunoglobulin Repertoire in Phage Lambda,” Science 246:1275-1281; andWard, et al. (1989) Nature 341:544-546. The polypeptides and antibodiesof the present invention may be used with or without modification,including chimeric or humanized antibodies. Frequently, the polypeptidesand antibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

[0110] The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.(1984) Meth. Enzymol. 104:3-55. Conversely, protein can be used fordepletion or cross absorptions to prepare selectively specific bindingcompositions.

[0111] Antibodies raised against each IL-B60 will also be useful toraise anti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

[0112] VI. Nucleic Acids

[0113] The described peptide sequences and the related reagents areuseful in detecting, isolating, or identifying a DNA clone encodingIL-B60, e.g., from a natural source. Typically, it will be useful inisolating a gene from a mammal, and similar procedures will be appliedto isolate genes from other species, e.g., warm blooded animals, such asbirds and mammals. Cross hybridization will allow isolation of IL-B60from the same, e.g., polymorphic variants, or other species. A number ofdifferent approaches will be available to successfully isolate asuitable nucleic acid clone.

[0114] The purified protein or polypeptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatemonoclonal or polyclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.

[0115] For example, a specific binding composition could be used forscreening of an expression library made from a cell line which expressesan IL-B60. Screening of intracellular expression can be performed byvarious staining or immunofluorescence procedures. Binding compositionscould be used to affinity purify or sort out cells expressing a surfacefusion protein.

[0116] The peptide segments can also be used to predict appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., SEQ ID NO: 1. In combination with polymerase chain reaction (PCR)techniques, synthetic oligonucleotides will be useful in selectingcorrect clones from a library. Complementary sequences will also be usedas probes, primers, or antisense strands. Various fragments should beparticularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

[0117] This invention contemplates use of isolated DNA or fragments toencode a biologically active corresponding IL-B60 polypeptide,particularly lacking the portion coding the untranslated portions of thedescribed sequence. In addition, this invention covers isolated orrecombinant DNA which encodes a biologically active protein orpolypeptide and which is capable of hybridizing under appropriateconditions with the DNA sequences described herein. Said biologicallyactive protein or polypeptide can be an intact antigen, or fragment, andhave an amino acid sequence disclosed in, e.g., SEQ ID NO: 2,particularly a mature, secreted-polypeptide. Further, this inventioncovers the use of isolated or recombinant DNA, or fragments thereof,which encode proteins which exhibit high identity to a secreted IL-B60.The isolated DNA can have the respective regulatory sequences in the 5′and 3′ flanks, e.g., promoters, enhancers, poly-A addition signals, andothers. Alternatively, expression may be effected by operably linking acoding segment to a heterologous promoter, e.g., by inserting a promoterupstream from an endogenous gene.

[0118] An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA,or a mixed polymer, which is substantially separated from othercomponents which naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Generally, the nucleic acid will be in a vector or fragment less thanabout 50 kb, usually less than about 30 kb, typically less than about 10kb, and preferably less than about 6 kb.

[0119] An isolated nucleic acid will generally be a homogeneouscomposition of molecules, but will, in some embodiments, contain minorheterogeneity. This heterogeneity is typically found at the polymer endsor portions not critical to a desired biological function or activity.

[0120] A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

[0121] Alternatively, it is performed to join together nucleic acidsegments of desired functions to generate a single genetic entitycomprising a desired combination of functions not found in the commonlyavailable natural forms. Restriction enzyme recognition sites are oftenthe target of such artificial manipulations, but other site specifictargets, e.g., promoters, DNA replication sites, regulation sequences,control sequences, or other useful features may be incorporated bydesign. A similar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesor polymorphic variants.

[0122] A significant “fragment” in a nucleic acid context is acontiguous segment of at least about 17 nucleotides, generally at leastabout 22 nucleotides, ordinarily at least about 29 nucleotides, moreoften at least about 35 nucleotides, typically at least about 41nucleotides, usually at least about 47 nucleotides, preferably at leastabout 55 nucleotides, and in particularly preferred embodiments will beat least about 60 or more nucleotides, e.g., 67, 73, 81, 89, 95, etc.

[0123] A DNA which codes for an IL-B60 protein will be particularlyuseful to identify genes, mRNA, and cDNA species which code for relatedor similar proteins; as well as DNAs which code for homologous proteinsfrom different species. There will be homologs in other species,including primates, rodents, canines, felines, and birds. Various IL-B60proteins should be homologous and are encompassed herein. However, evenproteins that have a more distant evolutionary relationship to theantigen can readily be isolated under appropriate conditions using thesesequences if they are sufficiently homologous. Primate IL-B60 proteinsare of particular interest.

[0124] Recombinant clones derived from the genomic sequences, e.g.,containing introns, will be useful for transgenic studies, including,e.g., transgenic cells and organisms, and for gene therapy. See, e.g.,Goodnow (1992) “Transgenic Animals” in Roitt (ed.) Encyclopedia ofImmunology, Academic Press, San Diego, pp. 1502-1504; Travis (1992)Science 256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi(1989) Science 244:1288; Robertson (ed. 1987) Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, IRL Press, Oxford; andRosenberg (1992) J. Clinical Oncology 10:180-199.

[0125] Substantial homology, e.g., identity, in the nucleic acidsequence comparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least about 58%,ordinarily at least about 65%, often at least about 71%, typically atleast about 77%, usually at least about 85%, preferably at least about95 to 98% or more, and in particular embodiments, as high as about 99%or more of the nucleotides. Alternatively, substantial homology existswhen the segments will hybridize under selective hybridizationconditions, to a strand, or its complement, typically using a sequenceof IL-B60, e.g., in SEQ ID NO: 1. Typically, selective hybridizationwill occur when there is at least about 55% identity over a stretch ofat least about 30 nucleotides, preferably at least about 75% over astretch of about 25 nucleotides, and most preferably at least about 90%over about 20 nucleotides. See, Kanehisa (1984) Nuc. Acids Res.12:203-213. The length of identity comparison, as described, may be overlonger stretches, and in certain embodiments will be over a stretch ofat least about 17 nucleotides, usually at least about 28 nucleotides,typically at least about 40 nucleotides, and preferably at least about75 to 100 or more nucleotides.

[0126] Stringent conditions, in referring to homology in thehybridization context, will be stringent combined conditions of salt,temperature, organic solvents, and other parameters, typically thosecontrolled in hybridization reactions. Stringent temperature conditionswill usually include temperatures in excess of about 30° C., usually inexcess of about 37° C., typically in excess of about 55° C., preferablyin excess of about 70° C. Stringent salt conditions will ordinarily beless than about 1000 mM, usually less than about 400 mM, typically lessthan about 250 mM, preferably less than about 150 mM, including about100, 50, or even 20 mM. However, the combination of parameters is muchmore important than the measure of any single parameter. See, e.g.,Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370. Hybridization understringent conditions should give background of at least 2-fold overbackground, preferably at least 3-5 or more.

[0127] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0128] Optical alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith and Waterman (1981) Adv.Appl. Math. 2:482, by the homology alignment algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methodof Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

[0129] One example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendrogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng and Doolittle(1987) J. Mol. Evol. 35:351-360. The method used is similar to themethod described by Higgins and Sharp (1989) CABIOS 5:151-153. Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

[0130] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described Altschul, et al. (1990) J. Mol. Biol.215:403-410. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http:www.ncbi.nlm.nih.gov/). This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul, et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extension of the word hits in each direction are halted when:the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or the end of either sequence is reached. The BLAST algorithm parametersW, T, and X determine the sensitivity and speed of the alignment. TheBLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62scoring matrix (see Henikoff and Henikoff (1989) Proc. Nat'l Acad. Sci.USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=4, anda comparison of both strands.

[0131] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

[0132] A further indication that two nucleic acid sequences ofpolypeptides are substantially identical is that the polypeptide encodedby the first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

[0133] IL-B60 from other mammalian species can be cloned and isolated bycross-species hybridization of closely related species. Homology may berelatively low between distantly related species, and thus hybridizationof relatively closely related species is advisable. Alternatively,preparation of an antibody preparation which exhibits less speciesspecificity may be useful in expression cloning approaches.

[0134] VII. Making IL-B60 or Complex; Mimetics

[0135] DNA which encodes the IL-B60 or fragments thereof can be obtainedby chemical synthesis, screening cDNA libraries, or screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170; Gublerand Hoffman (1983) Gene 25:263-269; and Glover (ed. 1984) DNA Cloning: APractical Approach, IRL Press, Oxford. Alternatively, the sequencesprovided herein provide useful PCR primers or allow synthetic or otherpreparation of suitable genes encoding an IL-B60; including naturallyoccurring embodiments.

[0136] This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length IL-B60 or fragments which can in turn, e.g.,be used to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies.

[0137] Vectors, as used herein, comprise plasmids, viruses,bacteriophage, integratable DNA fragments, and other vehicles whichenable the integration of DNA fragments into the genome of the host.See, e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: ALaboratory Manual, Elsevier, N.Y.; and Rodriguez, et al. (eds. 1988)Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Buttersworth, Boston, Mass.

[0138] For purposes of this invention, DNA sequences are operably linkedwhen they are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See, e.g., Rodriguez, et al., Chapter 10, pp. 205-236;Balbas and Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel,et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley,NY.

[0139] Representative examples of suitable expression vectors includepCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142;pMC1neo Poly-A, see Thomas, et al. (1987) Cell 51:503-512; and abaculovirus vector such as pAC 373 or pAC 610. See, e.g., Miller (1988)Ann. Rev. Microbiol. 42:177-199.

[0140] It will often be desired to express an IL-B60 polypeptide in asystem which provides a specific or defined glycosylation pattern. See,e.g., Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman(1990) Meth. Enzymol. 185:487-511.

[0141] The IL-B60, or a fragment thereof, may be engineered to bephosphatidyl inositol (PI) linked to a cell membrane, but can be removedfrom membranes by treatment with a phosphatidyl inositol cleavingenzyme, e.g., phosphatidyl inositol phospholipase-C. This releases theantigen in a biologically active form, and allows purification bystandard procedures of protein chemistry. See, e.g., Low (1989) Biochim.Biophys. Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; andBrunner, et al. (1991) J. Cell Biol. 114:1275-1283.

[0142] Now that the IL-B60 has been characterized, fragments orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis, Springer-Verlag, New York; Bodanszky (1984) ThePrinciples of Peptide Synthesis, Springer-Verlag, New York; andVillafranca (ed. 1991) Techniques in Protein Chemistry II, AcademicPress, San Diego, Calif.

[0143] VIII. Uses

[0144] The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in IL-B60mediated conditions, or below in the description of kits for diagnosis.The gene may be useful in forensic sciences, e.g., to distinguish rodentfrom human, or as a marker to distinguish between different cellsexhibiting differential expression or modification patterns. Theprovided compositions are useful reagents for, e.g., in vitro assays,scientific research, and the synthesis or manufacture of nucleic acids,polypeptides, or antibodies.

[0145] This invention also provides reagents with significant commercialand/or therapeutic potential. The IL-B60 (naturally occurring orrecombinant), fragments thereof, and antibodies thereto, along withcompounds identified as having binding affinity to IL-B60, should beuseful as reagents for teaching techniques of molecular biology,immunology, or physiology. Appropriate kits may be prepared with thereagents, e.g., in practical laboratory exercises in production or useof proteins, antibodies, cloning methods, histology, etc.

[0146] The reagents will also be useful in the treatment of conditionsassociated with abnormal physiology or development, includinginflammatory conditions. They may be useful in vitro tests for presenceor absence of interacting components, which may correlate with successof particular treatment strategies. In particular, modulation ofphysiology of various, e.g., hematopoietic or lymphoid, cells will beachieved by appropriate methods for treatment using the compositionsprovided herein. See, e.g., Thomson (1994; ed.) The Cytokine Handbook(2d ed.) Academic Press, San Diego; Metcalf and Nicola (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press; andAggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

[0147] For example, a disease or disorder associated with abnormalexpression or abnormal signaling by an IL-B60 should be a likely targetfor an agonist or antagonist. The new cytokine should play a role inregulation or development of hematopoietic cells, e.g., lymphoid cells,which affect immunological responses, e.g., inflammation and/orautoimmune disorders. Alternatively, it may affect vascular physiologyor development, or neuronal effects.

[0148] In particular, the cytokine should mediate, in various contexts,cytokine synthesis by the cells, proliferation, etc. Antagonists ofIL-B60, such as mutein variants of a naturally occurring form of IL-B60or blocking antibodies, may provide a selective and powerful way toblock immune responses, e.g., in situations as inflammatory orautoimmune responses. See also Samter, et al. (eds.) ImmunologicalDiseases vols. 1 and 2, Little, Brown and Co.

[0149] In addition, certain combination compositions would be useful,e.g., with other modulators of inflammation. Such other molecules mayinclude steroids, other versions of IL-6 and/or G-CSF, including speciesvariants, or viral homologs, and their respective antagonists.

[0150] Various abnormal conditions are known in each of the cell typesshown to produce IL-B60 mRNA by Northern blot analysis. See Berkow (ed.)The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.;Thorn, et al. Harrison's Principles of Internal Medicine, McGraw-Hill,N.Y.; and Weatherall, et al. (eds.) Oxford Textbook of Medicine, OxfordUniversity Press, Oxford. Many other medical conditions and diseasesinvolve activation by macrophages or monocytes, and many of these willbe responsive to treatment by an agonist or antagonist provided herein.See, e.g., Stites and Terr (eds.; 1991) Basic and Clinical ImmunologyAppleton and Lange, Norwalk, Conn.; and Samter, et al. (eds.)Immunological Diseases Little, Brown and Co. These problems should besusceptible to prevention or treatment using compositions providedherein. The pancreatic islet localization suggests a possible relevanceto diabetes.

[0151] IL-B60, antagonists, antibodies, etc., can be purified and thenadministered to a patient, veterinary or human. These reagents can becombined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers, excipients, or preservatives. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies orbinding fragments thereof, including forms which are not complementbinding.

[0152] Drug screening using IL-B60 or fragments thereof can be performedto identify compounds having binding affinity to or other relevantbiological effects on IL-B60 functions, including isolation ofassociated components. Subsequent biological assays can then be utilizedto determine if the compound has intrinsic stimulating activity and istherefore a blocker or antagonist in that it blocks the activity of thecytokine. Likewise, a compound having intrinsic stimulating activity canactivate the signal pathway and is thus an agonist in that it simulatesthe activity of IL-B60. This invention further contemplates thetherapeutic use of blocking antibodies to IL-B60 as antagonists and ofstimulatory antibodies as agonists. This approach should be particularlyuseful with other IL-B60 species variants.

[0153] The quantities of reagents necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer (1990) Science249:1527-1533.

[0154] IL-B60, fragments thereof, and antibodies to it or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, topical, or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. See, e.g., Gilman, et al.(eds. 1990) Goodman and Gilman's: The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn.; Avis, etal. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York; Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Tablets, Dekker, New York; and Lieberman, et al. (eds. 1990)Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York. Thetherapy of this invention may be combined with or used in associationwith other agents, e.g., other cytokines, including IL-6 or G-CSF, ortheir respective antagonists.

[0155] Both naturally occurring and recombinant forms of the IL-B60s ofthis invention are particularly useful in kits and assay methods whichare capable of screening compounds for binding activity to the proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds in a shortperiod. See, e.g., Fodor, et al. (1991) Science 251:767-773, whichdescribes means for testing of binding affinity by a plurality ofdefined polymers synthesized on a solid substrate. The development ofsuitable assays can be greatly facilitated by the availability of largeamounts of purified, soluble IL-B60 as provided by this invention.

[0156] Other methods can be used to determine the critical residues inIL-B60-IL-B60 receptor interactions. Mutational analysis can beperformed, e.g., see Somoza, et al. (1993) J. Expt1. Med. 178:549-558,to determine specific residues critical in the interaction and/orsignaling. PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (Kingand Sternberg (1996) Protein Sci. 5:2298-2310) can provide secondarystructure predictions of α-helix (H), β-strand (E), or coil (L). HelicesA and D are most important in receptor interaction, with the D helix themore important region. See Table 2.

[0157] For example, antagonists can normally be found once the antigenhas been structurally defined, e.g., by tertiary structure data. Testingof potential interacting analogs is now possible upon the development ofhighly automated assay methods using a purified IL-B60. In particular,new agonists and antagonists will be discovered by using screeningtechniques described herein. Of particular importance are compoundsfound to have a combined binding affinity for a spectrum of IL-B60molecules, e.g., compounds which can serve as antagonists for speciesvariants of IL-B60.

[0158] One method of drug screening utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant DNA moleculesexpressing an IL-B60. Cells may be isolated which express an IL-B60 inisolation from other molecules. Such cells, either in viable or fixedform, can be used for standard binding partner binding assays. See also,Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990)Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses.

[0159] Another technique for drug screening involves an approach whichprovides high throughput screening for compounds having suitable bindingaffinity to an IL-B60 and is described in detail in Geysen, EuropeanPatent Application 84/03564, published on Sep. 13, 1984. First, largenumbers of different small peptide test compounds are synthesized on asolid substrate, e.g., plastic pins or some other appropriate surface,see Fodor, et al. (1991). Then all the pins are reacted withsolubilized, unpurified or solubilized, purified IL-B60, and washed. Thenext step involves detecting bound IL-B60.

[0160] Rational drug design may also be based upon structural studies ofthe molecular shapes of the IL-B60 and other effectors or analogs.Effectors may be other proteins which mediate other functions inresponse to binding, or other proteins which normally interact withIL-B60, e.g., a receptor. One means for determining which sites interactwith specific other proteins is a physical structure determination,e.g., x-ray crystallography or 2 dimensional NMR techniques. These willprovide guidance as to which amino acid residues form molecular contactregions, as modeled, e.g., against other cytokine-receptor models. For adetailed description of protein structural determination, see, e.g.,Blundell and Johnson (1976) Protein Crystallography, Academic Press, NewYork.

[0161] IX. Kits

[0162] This invention also contemplates use of IL-B60 proteins,fragments thereof, peptides, and their fusion products in a variety ofdiagnostic kits and methods for detecting the presence of another IL-B60or binding partner. Typically the kit will have a compartment containingeither a defined IL-B60 peptide or gene segment or a reagent whichrecognizes one or the other, e.g., IL-B60 fragments or antibodies.

[0163] A kit for determining the binding affinity of a test compound toan IL-B60 would typically comprise a test compound; a labeled compound,for example a binding partner or antibody having known binding affinityfor IL-B60; a source of IL-B60 (naturally occurring or recombinant); anda means for separating bound from free labeled compound, such as a solidphase for immobilizing the molecule. Once compounds are screened, thosehaving suitable binding affinity to the antigen can be evaluated insuitable biological assays, as are well known in the art, to determinewhether they act as agonists or antagonists to the IL-B60 signalingpathway. The availability of recombinant IL-B60 polypeptides alsoprovide well defined standards for calibrating such assays.

[0164] A preferred kit for determining the concentration of, e.g., anIL-B60 in a sample would typically comprise a labeled compound, e.g.,binding partner or antibody, having known binding affinity for theantigen, a source of cytokine (naturally occurring or recombinant) and ameans for separating the bound from free labeled compound, e.g., a solidphase for immobilizing the IL-B60. Compartments containing reagents, andinstructions, will normally be provided.

[0165] Antibodies, including antigen binding fragments, specific for theIL-B60 or fragments are useful in diagnostic applications to detect thepresence of elevated levels of IL-B60 and/or its fragments. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the antigen in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-binding partner complex) or heterogeneous (witha separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525; Harlow andLane (1980) Antibodies: A Laboratory Manual, CSH Press, NY; and Coligan,et al. (eds. 1993) Current Protocols in Immunology, Greene and Wiley,NY.

[0166] Anti-idiotypic antibodies may have similar use to diagnosepresence of antibodies against an IL-B60, as such may be diagnostic ofvarious abnormal states. For example, overproduction of IL-B60 mayresult in production of various immunological reactions which may bediagnostic of abnormal physiological states, particularly inproliferative cell conditions such as cancer or abnormal activation ordifferentiation. Moreover, the distribution pattern available providesinformation that the cytokine is expressed in pancreatic islets,suggesting the possibility that the cytokine may be involved in functionof that organ, e.g., in a diabetes relevant medical condition.

[0167] Frequently, the reagents for diagnostic assays are supplied inkits, so as to optimize the sensitivity of the assay. For the subjectinvention, depending upon the nature of the assay, the protocol, and thelabel, either labeled or unlabeled antibody or binding partner, orlabeled IL-B60 is provided. This is usually in conjunction with otheradditives, such as buffers, stabilizers, materials necessary for signalproduction such as substrates for enzymes, and the like. Preferably, thekit will also contain instructions for proper use and disposal of thecontents after use. Typically the kit has compartments for each usefulreagent. Desirably, the reagents are provided as a dry lyophilizedpowder, where the reagents may be reconstituted in an aqueous mediumproviding appropriate concentrations of reagents for performing theassay.

[0168] Many of the aforementioned constituents of the drug screening andthe diagnostic assays may be used without modification or may bemodified in a variety of ways. For example, labeling may be achieved bycovalently or non-covalently joining a moiety which directly orindirectly provides a detectable signal. In any of these assays, thebinding partner, test compound, IL-B60, or antibodies thereto can belabeled either directly or indirectly. Possibilities for direct labelinginclude label groups: radiolabels such as ¹²⁵I, enzymes such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

[0169] There are also numerous methods of separating the bound from thefree IL-B60, or alternatively the bound from the free test compound. TheIL-B60 can be immobilized on various matrixes followed by washing.Suitable matrixes include plastic such as an ELISA plate, filters, andbeads. See, e.g., Coligan, et al. (eds.. 1993) Current Protocols inImmunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle, et al. (1984)Clin. Chem. 30:1457-1461, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,678.

[0170] Methods for linking proteins or their fragments to the variouslabels have been extensively reported in the literature and do notrequire detailed discussion here. Many of the techniques involve the useof activated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

[0171] Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan IL-B60. These sequences can be used as probes for detecting levels ofthe IL-B60 message in samples from patients suspected of having anabnormal condition, e.g., inflammatory or autoimmune. Since the cytokinemay be a marker or mediator for activation, it may be useful todetermine the numbers of activated cells to determine, e.g., whenadditional therapy may be called for, e.g., in a preventative fashionbefore the effects become and progress to significance. The preparationof both RNA and DNA nucleotide sequences, the labeling of the sequences,and the preferred size of the sequences has received ample descriptionand discussion in the literature. See, e.g., Langer-Safer, et al. (1982)Proc. Nat'l. Acad. Sci. 79:4381-4385; Caskey (1987) Science 236:962-967;and Wilchek et al. (1988) Anal. Biochem. 171:1-32.

[0172] Diagnostic kits which also test for the qualitative orquantitative expression of other molecules are also contemplated.Diagnosis or prognosis may depend on the combination of multipleindications used as markers. Thus, kits may test for combinations ofmarkers. See, e.g., Viallet, et al. (1989) Progress in Growth FactorRes. 1:89-97. Other kits may be used to evaluate other cell subsets.

[0173] X. Isolating a IL-B60 Receptor

[0174] Having isolated a ligand of a specific ligand-receptorinteraction, methods exist for isolating the receptor. See, Gearing, etal. (1989) EMBO J. 8:3667-3676. For example, means to label the IL-B60cytokine without interfering with the binding to its receptor can bedetermined. For example, an affinity label can be fused to either theamino- or carboxyl-terminus of the ligand. Such label may be a FLAGepitope tag, or, e.g., an Ig or Fc domain. An expression library can bescreened for specific binding of the cytokine, e.g., by cell sorting, orother screening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA90:11267-11271; and Liu, et al. (1994) J. Immunol. 152:1821-29.Alternatively, a panning method may be used. See, e.g., Seed and Aruffo(1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369.

[0175] Protein cross-linking techniques with label can be applied toisolate binding partners of the IL-B60 cytokine. This would allowidentification of proteins which specifically interact with thecytokine, e.g., in a ligand-receptor like manner.

[0176] Early experiments will be performed to determine whether theknown IL-6 or G-CSF receptor components are involved in response(s) toIL-B60. It is also quite possible that these functional receptorcomplexes may share many or all components with an IL-B60 receptorcomplex, either a specific receptor subunit or an accessory receptorsubunit.

[0177] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

EXAMPLES

[0178] I. General Methods

[0179] Many of the standard methods below are described or referenced,e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory ManualCold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook,et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3,CSH Press, NY; Ausubel, et al. Biology Greene Publishing Associates,Brooklyn, N.Y.; Ausubel, et al. (1987 and Supplements) Current Protocolsin Molecular Biology Wiley/Greene, NY.; Innis, et al. (eds. 1990) PCRProtocols: A Guide to Methods and Applications Academic Press, NY;Bonifacino, et al. Current Protocols in Cell Biology Wiley, NY.; andDoyle, et al. Cell and Tissue Culture: Laboratory Protocols Wiley, NY.Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; Coligan, et al. (1995 and supplements) Current Protocols inProtein Science John Wiley and Sons, New York, N.Y.; Matsudaira (ed.1993) A Practical Guide to Protein and Peptide Purification forMicrosequencing, Academic Press, San Diego, Calif.; and manufacturer'sliterature on use of protein purification products, e.g., Pharmacia,Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination withrecombinant techniques allow fusion to appropriate segments (epitopetags), e.g., to a FLAG sequence or an equivalent which can be fused,e.g., via a protease-removable sequence. See, e.g., Hochuli (1990)“Purification of Recombinant Proteins with Metal Chelate Absorbent” inSetlow (ed.) Genetic Engineering, Principle and Methods 12:87-98, PlenumPress, NY; and Crowe, et al. (1992) QIAexpress: The High LevelExpression & Protein Purification System QUIAGEN, Inc., Chatsworth,Calif.

[0180] Computer sequence analysis is performed, e.g., using availablesoftware programs, including those from the University of WisconsinGenetics Computer Group (GCG), Madison, Wis., the NCBI at NIH, andGenBank, NCBI, EMBO, and other sources of public sequence. Otheranalysis sources include, e.g., RASMOL program, see Bazan, et al. (1996)Nature 379:591; Lodi, et al. (1994) Science 263:1762-1766; Sayle andMilner-White (1995) TIBS 20:374-376; and Gronenberg, et al. (1991)Protein Engineering 4:263-269; and DSC, see King and Sternberg (1996)Protein Sci. 5:2298-2310. See, also, Wilkins, et al. (eds. 1997)Proteome Research: New Frontiers in Functional Genomics Springer-Verlag,NY.; Salzberg, et al. (eds. 1998) Computational Methods in MolecularBiology Elsevier, NY.; and Birren, et al. (eds. 1997) Genome Analysis: ALaboratory Manual Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0181] Standard immunological techniques are described, e.g., inHertzenberg, et al. (eds. 1996) Weir's Handbook of ExperimentalImmunology vols. 1-4, Blackwell Science; Coligan (1991 and updates)Current Protocols in Immunology Wiley/Greene, NY.; and Methods inEnzymology vols. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162,and 163. Cytokine assays are described, e.g., in Thomson (ed. 1994) TheCytokine Handbook (2d ed.) Academic Press, San Diego; Metcalf and Nicola(1995) The Hematopoietic Colony Stimulating Factors Cambridge UniversityPress; and Aggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

[0182] Assays for vascular biological activities are well known in theart. They will cover angiogenic and angiostatic activities in tumor, orother tissues, e.g., arterial smooth muscle proliferation (see, e.g.,Koyoma, et al. (1996) Cell 87:1069-1078), monocyte adhesion to vascularepithelium (see McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc.See also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am.J. Pathol. 147:668-677; Thyberg, et al. (1990) Atherosclerosis10:966-990; and Gumbiner (1996) Cell 84:345-357.

[0183] Assays for neural cell biological activities are described, e.g.,in Wouterlood (ed. 1995) Neuroscience Protocols modules 10, Elsevier;Methods in Neurosciences Academic Press; and Neuromethods Humana Press,Totowa, N.J. Methodology of developmental systems is described, e.g., inMeisami (ed.) Handbook of Human Growth and Developmental Biology CRCPress; and Chrispeels (ed.) Molecular Techniques and Approaches inDevelopmental Biology Interscience.

[0184] FACS analyses are described in Melamed, et al. (1990) FlowCytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al.(1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.

[0185] II. Cloning of Human IL-B60

[0186] The sequence of the gene is provided in Table 1. The sequence isderived from a genomic human sequence. These sequences allow preparationof PCR primers, or probes, to determine cellular distribution of thegene. The sequences allow isolation of genomic DNA which encode themessage.

[0187] Using the probe or PCR primers, various tissues or cell types areprobed to determine cellular distribution. PCR products are clonedusing, e.g., a TA cloning kit (Invitrogen). The resulting cDNA plasmidsare sequenced from both termini on an automated sequencer (AppliedBiosystems).

[0188] III. Cellular Expression of IL-B60

[0189] An appropriate probe or primers specific for cDNA encodingprimate IL-B60 are prepared. Typically, the probe is labeled, e.g., byrandom priming. The expression is probably in the cell types described,and perhaps also in pancreatic islets.

[0190] The presence of a leader sequence led to the expectation offinding IL-B60 secreted when expressed in mammalian cells. Transfectionof 293T cells with a tagged form of hIL-B60 (hIL-B60-Etag) did notresult in efficient secretion of IL-B60. Instead, IL-B60 could only beimmunoprecipitated from the lysate of the transfected cells. Thepossibility was investigated of IL-B60 being a composite factor likeIL-12 (p35/p40) and thus needing a partner for secretion. Among thenon-signaling receptors of the IL-6 family the recently described and,thus far, orphan, receptor CLF-1 (NR6) also showed high level ofhomology between human and murine forms (>95% amino acid identity).Based on these observations a hypothesis was generated that the IL-B60and CLF-1 are partners.

[0191] Southern Analysis: DNA (5 μg) from a primary amplified cDNAlibrary is digested with appropriate restriction enzymes to release theinserts, run on a 1% agarose gel and transferred to a nylon membrane(Schleicher and Schuell, Keene, N.H.).

[0192] Samples for human mRNA isolation can include, e.g., peripheralblood mononuclear cells (monocytes, T cells, NK cells, granulocytes, Bcells), resting (T100); peripheral blood mononuclear cells, activatedwith anti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72,resting (T102); T cell, TH0 clone Mot 72, activated with anti-CD28 andanti-CD3 for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergictreated with specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1clone HY06, resting (T107); T cell, TH1 clone HY06, activated withanti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 cloneHY06, anergic treated with specific peptide for 2, 6, 12 h pooled(T109); T cell, TH2 clone HY935, resting (T110); T cell, TH2 cloneHY935, activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled(T111); T cell tumor lines Jurkat and Hut78., resting (T117); T cellclones, pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting(T118); T cell random γδ T cell clones, resting (T119); CD28-T cellclone; Splenocytes, resting (B100); Splenocytes, activated withanti-CD40 and IL-4 (B101); B cell EBV lines pooled WT49, RSB, JY, CVIR,721.221, RM3, HSY, resting (B102); B cell line JY, activated with PMAand ionomycin for 1, 6 h pooled (B103); NK 20 clones pooled, resting(K100); NK 20 clones pooled, activated with PMA and ionomycin for 6 h(K101); NKL clone, derived from peripheral blood of LGL leukemiapatient, IL-2 treated (K106); hematopoietic precursor line TF1,activated with PMA and ionomycin for 1, 6 h pooled (C100); U937premonocytic line, resting (M100); U937 premonocytic line, activatedwith PMA and ionomycin for 1, 6 h pooled (M101); elutriated monocytes,activated with LPS, IFNγ, anti-IL-10 for 1, 2, 6, 12, 24 h pooled(M102); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 1, 2,6, 12, 24 h pooled (M103); elutriated monocytes, activated with LPS,IFNγ, anti-IL-10 for 4, 16 h pooled (M106); elutriated monocytes,activated with LPS, IFNγ, IL-10 for 4, 16 h pooled (M107); elutriatedmonocytes, activated LPS for 1 h (M108); elutriated monocytes, activatedLPS for 6 h (M109); DC 70% CD1a+, from CD34+GM-CSF, TNFα 12 days,resting (D101); DC 70% CD1a+, from CD34+GM-CSF, TNFα 12 days, activatedwith PMA and ionomycin for 1 hr (D102); DC 70% CD1a+, from CD34+GM-CSF,TNFα 12 days, activated with PMA and ionomycin for 6 hr (D103); DC 95%CD1a+, from CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMAand ionomycin for 1, 6 h pooled (D104); DC 95% CD14+, ex CD34+ GM-CSF,TNFα 12 days FACS sorted, activated with PMA and ionomycin 1, 6 hrpooled (D105); DC CD1a+ CD86+, from CD34+ GM-CSF, TNFα 12 days FACSsorted, activated with PMA and ionomycin for 1, 6 h pooled (D106); DCfrom monocytes GM-CSF, IL-4 5 days, resting (D107); DC from monocytesGM-CSF, IL-4 5 days, resting (D108); DC from monocytes GM-CSF, IL-4 5days, activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF,IL-4 5 days, activated TNFα, monocyte supe for 4, 16 h pooled (D110);epithelial cells, unstimulated; epithelial cells, IL-1β activated; lungfibroblast sarcoma line MRC5, activated with PMA and ionomycin for 1, 6h pooled (C101); kidney epithelial carcinoma cell line CHA, activatedwith PMA and ionomycin for 1, 6 h pooled (C102). Expression of IL-B60transcript was very high in elutriated monocytes, activated with LPS,IFNγ, anti-IL-10 for 4, 16 h pooled (M106); elutriated monocytes,activated with LPS, IFNγ, anti-IL-10 for 1, 2, 6, 12, 24 h pooled(M102); elutriated monocytes, activated LPS for 6 h (M109); andelutriated monocytes, activated LPS for 1 h (M108).

[0193] Samples for mouse mRNA expression can include, e.g., restingmouse fibroblastic L cell line (C200); Braf:ER (Braf fusion to estrogenreceptor) transfected cells, control (C201); Mel14+ naive T cells fromspleen, resting (T209); Mel14+ naive T cells from spleen, stimulatedwith IFNγ, IL-12, and anti IL-4 to polarize to TH1 cells, exposed toIFNγ and IL-4 for 6, 12, 24 h, pooled (T210); Mel14+ naive T cells fromspleen, stimulated with IL-4 and anti IFNγ to polarize to Th2 cells,exposed to IL-4 and anti IFNγ for 6, 13, 24 h, pooled (T211); T cells,TH1 polarized (Mel14 bright, CD4+ cells from spleen, polarized for 7days with IFN-γ and anti IL-4; T200); T cells, TH2 polarized (Mel14bright, CD4+ cells from spleen, polarized for 7 days with IL-4 andanti-IFN-γ; T201); T cells, highly TH1 polarized 3× from transgenicBalb/C (see Openshaw; et al. (1995) J. Exp. Med. 182:1357-1367;activated with anti-CD3 for 2, 6, 24 h pooled; T202); T cells, highlyTH2 polarized 3× from transgenic Balb/C (activated with anti-CD3 for 2,6, 24 h pooled (T203); T cells, highly TH1 polarized 3× from transgenicC57 bl/6 (activated with anti-CD3 for 2, 6, 24 h pooled; T212); T cells,highly TH2 polarized 3× from transgenic C57 bl/6 (activated withanti-CD3 for 2, 6, 24 h pooled; T213); T cells, highly TH1 polarized(naive CD4+ T cells from transgenic Balb/C, polarized 3× with IFNγ,IL-12, and anti-IL-4; stimulated with IGIF, IL-12, and anti IL-4 for 6,12, 24 h, pooled); CD44-CD25+ pre T cells, sorted from thymus (T204);TH1 T cell clone D1.1, resting for 3 weeks after last stimulation withantigen (T205); TH1 T cell clone D1.1 , 10 μg/ml ConA stimulated 15 h(T206); TH2 T cell clone CDC35, resting for 3 weeks after laststimulation with antigen (T207); TH2 T cell clone CDC35, 10 μg/ml ConAstimulated 15 h (T208); unstimulated B cell line CH12 (B201);unstimulated mature B cell leukemia cell line A20 (B200); unstimulatedlarge B cells from spleen (B202); B cells from total spleen, LPSactivated (B203); metrizamide enriched dendritic cells from spleen,resting (D200); dendritic cells from bone marrow, resting (D201);unstimulated bone marrow derived dendritic cells depleted with antiB220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4 (D202);bone marrow derived dendritic cells depleted with anti B220, anti CD3,and anti Class II, cultured in GM-CSF and IL-4, stimulated with antiCD40 for 1, 5 d, pooled (D203); monocyte cell line RAW 264.7 activatedwith LPS 4 h (M200); bone-marrow macrophages derived with GM and M-CSF(M201); bone-marrow macrophages derived with GM-CSF, stimulated withLPS, IFNγ, and IL-10 for 24 h (M205); bone-marrow macrophages derivedwith GM-CSF, stimulated with LPS, IFNγ, and anti IL-10 for 24 h (M206);peritoneal macrophages (M207); macrophage cell line J774, resting(M202); macrophage cell line J774+LPS+anti-IL-10 at 0.5, 1, 3, 6, 12 hpooled (M203); macrophage cell line J774+LPS+IL-10 at 0.5, 1, 3, 5, 12 hpooled (M204); unstimulated mast cell lines MC-9 and MCP-12 (M208);immortalized endothelial cell line derived from brain microvascularendothelial cells, unstimulated (E200); immortalized endothelial cellline derived from brain microvascular endothelial cells, stimulatedovernight with TNFα (E201); immortalized endothelial cell line derivedfrom brain microvascular endothelial cells, stimulated overnight withTNFα (E202); immortalized endothelial cell line derived from brainmicrovascular endothelial cells, stimulated overnight with TNFα andIL-10 (E203); total aorta from wt C57 bl/6 mouse; total aorta from 5month ApoE KO mouse (X207); total aorta from 12 month ApoE KO mouse(X207); wt thymus (0214); total thymus, rag-1 (0208); total kidney,rag-1 (0209); total kidney, NZ B/W mouse; and total heart, rag-1 (0202).

[0194] The human IL-B60 was found expressed in T cells; the Th0 cloneMot72 (activated); activated PBL; monocytes; dendritic cells; fetallung, and heavy smoker lung samples.

[0195] The CLF-1 was found expressed in dendritic cells; splenocytes;Th1 cells; fetal lung; and lung samples. This distribution is consistentwith the complex being important in immune function, e.g., dendritic andimmune cells, and in lung physiology.

[0196] Since CLF-1 is necessary for IL-B60 secretion in vitro, varioushuman and mouse cDNA libraries were screened for co-expression of bothmRNAs. Highest expression for both was found in adult human splenocytes,T cells, activated monocytes and dendritic cells and in fetal lung, anduterus. In mouse libraries, co-expression was strongest in adult lung.

[0197] IV. Chromosome Mapping of IL-B60

[0198] An isolated cDNA encoding the IL-B60 is used. Chromosome mappingis a standard technique. See, e.g., BIOS Laboratories (New Haven, Conn.)and methods for using a mouse somatic cell hybrid panel with PCR.

[0199] The human IL-B60 gene has been localized to human chromosome 11.

[0200] V. Purification of IL-B60 Protein or Complexes

[0201] Multiple transfected cell lines are screened for one whichexpresses the cytokine at a high level compared with other cells.Alternatively, a recombinant construct with both subunits can be made.Various cell lines are screened and selected for their favorableproperties in handling. Natural IL-B60 can be isolated from naturalsources, or by expression from a transformed cell using an appropriateexpression vector. Purification of the expressed protein or complex isachieved by standard procedures, or may be combined with engineeredmeans for effective purification at high efficiency from cell lysates orsupernatants. FLAG or His6 segments can be used for such purificationfeatures. Alternatively, affinity chromatography may be used withspecific antibodies, see below.

[0202] Protein is produced in coli, insect cell, or mammalian expressionsystems, as desired.

[0203] VI. Isolation of Homologous IL-B60 Genes

[0204] The IL-B60 cDNA, or other species counterpart sequence, can beused as a hybridization probe to screen a library from a desired source,e.g., a primate cell cDNA library. Many different species can bescreened both for stringency necessary for easy hybridization, and forpresence using a probe. Appropriate hybridization conditions will beused to select for clones exhibiting specificity of cross hybridization.

[0205] Screening by hybridization using degenerate probes based upon thepeptide sequences will also allow isolation of appropriate clones.Alternatively, use of appropriate primers for PCR screening will yieldenrichment of appropriate nucleic acid clones.

[0206] Similar methods are applicable to isolate either species,polymorphic, or allelic variants. Species variants are isolated usingcross-species hybridization techniques based upon isolation of a fulllength isolate or fragment from one species as a probe.

[0207] Alternatively, antibodies raised against human IL-B60 will beused to screen for cells which express cross-reactive proteins from anappropriate, e.g., cDNA library. The purified protein or definedpeptides are useful for generating antibodies by standard methods, asdescribed above. Synthetic peptides or purified protein are presented toan immune system to generate monoclonal or polyclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. The resulting antibodies are used for screening,purification, or diagnosis, as described.

[0208] VII. Antibodies Specific for IL-B60 or Complexes

[0209] Synthetic peptides or purified protein are presented to an immunesystem to generate monoclonal or polyclonal antibodies. See, e.g.,Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlowand Lane (1989) Antibodies: A Laboratory Manual Cold Spring HarborPress. Polyclonal serum, or hybridomas may be prepared. In appropriatesituations, the binding reagent is either labeled as described above,e.g., fluorescence or otherwise, or immobilized to a substrate forpanning methods. Immunoselection, immunodepletion, and relatedtechniques are available to prepare selective reagents, as desired,e.g., for the IL-B60 alone, or the complex between the two subunits.

[0210] VIII. IL-B60 and CLF-1 Coprecipitate

[0211] A CLF-1-FLAG construct was prepared in an expression vector. AnIL-B60Etag (epitope tagged) construct was also prepared. Transienttransfection into COS cells either with the IL-B60Etag construct alone,the CLF-1-FLAG construct alone, or both together. Cells were labeledwith ³⁵S methionine. The supernatants and cells were collected.

[0212] Upon co-transfection of cells with IL-B60-Etag and solublereceptor CLF-1-Flag, secretion of both ligand and soluble receptor wasgreatly enhanced. Both could be immunoprecipitated with antibodiesagainst either the ligand (anti Etag) or the receptor (anti Flag),indicating that IL-B60 and CLF-1 form a soluble cytokine/receptorcomplex similar to IL-12 (p35/p40). See Gubler, et al. (1991) Proc.Nat'l Acad. Sci. USA 88:4143-4147; Wolf, et al. (1991) J. Immunol.146:3074-3081. Thus, coexpression with a correct partner will result ina dramatic increase in the secretion of the gene products. Coexpressionof IL-B60 with other soluble receptors including Ebi3 (Devergne, et al.(1996) J. Virol. 70:1143-1153), IL-12 p40, and sCNTFR (Davis, et al.(1991) Science 253:59-63) did not result in efficient secretion of theligand.

[0213] The supernatants were immunoprecipitated with either anti-FLAG M2(precipitates CLF-1) or anti-Etag Ab (precipitates IL-B60). InIL-B60Etag transfectants alone, the level of expression in thesupernatant detected using the antiEtag antibodies was very low. Incontrast, in the double transfectants, the IL-B60Etag and a secondlabeled band were immunoprecipitated. The second band corresponds to theCLF-1. Thus, the Etag antibody immunoprecipitates both proteins, e.g.,they form a complex. In the single transfectant CLF-1FLAG, a little bitof CLF-1FLAG protein is immunoprecipitated with the anti-FLAG M2 Ab.This result is consistent with the other soluble receptors, e.g., forp40 component of IL-12. However, in the double transfectants not only ismore CLF-1 seen, but now also IL-B60. The immunoprecipitation works inboth directions.

[0214] IX. IL-B60 Binds to the CNTFR

[0215] To identify the signaling receptors for IL-B60/CLF-1 conditionedmedium from hIL-B60 and mCLF-1 cotransfected 293T cells was added toBA/F3 cells stably transfected with human gp130 alone or hgp130 incombination with the hIL-6R, hOSMR, hLIFR, or hLIFR and hCNTFR,respectively. Only BA/F3 cells expressing gp130, LIFR, and CNTFR showeda proliferative response upon stimulation with IL-B60/CLF-1. To analyzethe possibility of a signaling complex consisting of CNTFR/gp130 orCNTFR/LIFR only, two soluble fusion proteins were designed connectingeither the CNTFR or CLF-1 to IL-B60 via a flexible linker. Similarso-called hyper-cytokines have been shown to be 100-1000× more active oncells than cytokine and soluble receptor added separately. See Fischer,et al. (1997) Nature Biotechnol. 15:142-145. Hyper-CNTFR-IL-B60 was ableto induce proliferation of BAF3/gp130/LIFR cells but not of BAF3/gp130cells, showing that the LIFR is a component of the signaling complex.Stimulation of cells with hyper-CLF-1-IL-B60 did not result inproliferation of any cell line. This indicated that although necessaryfor IL-B60 secretion, CLF-1 is not a subunit of the active signalingreceptor complex.

[0216] Involvement of gp130 in the active receptor complex was shownwith a neutralizing antibody against gp130 which completely blocked thisresponse. Furthermore, analysis of signal transducers in lysates fromBA/F3 cells expressing gp130, LIFR, and CNTFR showed that STAT3 is onlyphosphorylated after stimulation with either co-expressed IL-B60 andCLF-1 or with the CNTFR-IL-B60 fusion protein but not with theCLF-1-IL-B60 fusion.

[0217] X. Evaluation of Breadth of Biological Functions

[0218] Biological activities of IL-B60 or complex are tested based,e.g., on the sequence and structural homology between IL-B60 and IL-6and G-CSF. Initially, assays that had shown biological activities ofIL-6 or G-CSF are examined.

[0219] A. Regulation of IL-B60 and CLF-1 After Sciatic Nerve Injury

[0220] IL-B60 and CLF-1 expression in the mouse spinal cord was analyzedin unilateral transection of the sciatic nerve followed by separation ofproximal and distal nerve stumps, thus preventing regeneration. Atvarious time points, tissue from the transection area was collected andanalyzed by quantitative PCR for expression of IL-B60 and CLF-1.Transection of the sciatic nerve resulted in fast and long lastingupregulation of ligand and receptor. After 6 hrs IL-B60 and CLF-1 wereupregulated. Expression was still elevated 20 days after transectionwhen compared to non-lesioned or sham-lesioned nerves. In regeneratingaxons (crushed nerves) both IL-B60 and CLF-1 are downregulated after 12h, but whereas IL-B60 expression almost reaches levels of non-lesionednerves after 20 days, CLF-1 levels peak after 20 days. This might pointto an additional function of CLF-1, possibly in remyelination, whichstarts after two weeks. Transection of the sciatic nerve in mice lackingGM-CSF and a macrophage response in nerve shows that IL-B60 expressionafter 4 days is not altered compared to normal mice. However, CLF-1levels in those mice are heterogeneous, with a range from no alterationto an almost 4 fold increase of expression compared to normallittermates.

[0221] B. Effects on Proliferation of Cells

[0222] The effect on proliferation of various cell types are evaluatedwith various concentrations of cytokine. A dose response analysis isperformed, in combinations with the related cytokines IL-6, G-CSF, etc.A cytosensor machine may be used, which detects cell metabolism andgrowth (Molecular Devices, Sunnyvale, Calif.).

[0223] C. Effects on the Expression of Cell Surface Molecules on HumanMonocytes

[0224] Monocytes are purified by negative selection from peripheralblood mononuclear cells of normal healthy donors. Briefly, 3×10⁸ ficollbanded mononuclear cells are incubated on ice with a cocktail ofmonoclonal antibodies (Becton-Dickinson; Mountain View, Calif.)consisting, e.g., of 200 μl of αCD2 (Leu-5A), 200 μl of αCD3 (Leu-4),100 μl of αCD8 (Leu 2a), 100 μl of αCD19 (Leu-12), 100 μl of αCD20(Leu-16), 100 μl of αCD56 (Leu-19), 100 μl of αCD67 (IOM 67; Immunotech,Westbrook, Me.), and anti-glycophorin antibody (10F7MN, ATCC, Rockville,Md.). Antibody bound cells are washed and then incubated with sheepanti-mouse IgG coupled magnetic beads (Dynal, Oslo, Norway) at a bead tocell ratio of 20:1. Antibody bound cells are separated from monocytes byapplication of a magnetic field. Subsequently, human monocytes arecultured in Yssel's medium (Gemini Bioproducts, Calabasas, Calif.)containing 1% human AB serum in the absence or presence of IL-B60, IL-6,G-CSF or combinations.

[0225] Analyses of the expression of cell surface molecules can beperformed by direct immunofluorescence. For example, 2×10⁵ purifiedhuman monocytes are incubated in phosphate buffered saline (PBS)containing 1% human serum on ice for 20 minutes. Cells are pelleted at200×g. Cells are resuspended in 20 ml PE or FITC labeled mAb. Followingan additional 20 minute incubation on ice, cells are washed in PBScontaining 1% human serum followed by two washes in PBS alone. Cells arefixed in PBS containing 1% paraformaldehyde and analyzed on FACScan flowcytometer (Becton Dickinson; Mountain View, Calif.). Exemplary mAbs areused, e.g.: CD11b (anti-mac1), CD11c (a gp150/95), CD14 (Leu-M3), CD54(Leu 54), CD80 (anti-BB1/B7), HLA-DR (L243) from Becton-Dickinson andCD86 (FUN 1; Pharmingen), CD64 (32.2; Medarex), CD40 (mAb89;Schering-Plough France).

[0226] D. Effects of IL-B60 or Complex on Cytokine Production by HumanMonocytes

[0227] Human monocytes are isolated as described and cultured in Yssel'smedium (Gemini Bioproducts, Calabasas, Calif.) containing 1% human ABserum in the absence or presence of IL-B60 (1/100 dilution baculovirusexpressed material). In addition, monocytes are stimulated with LPS (E.coli 0127:B8 Difco) in the absence or presence of IL-B60 and theconcentration of cytokines (IL-1β, IL-6, TNFα, GM-CSF, and IL-10) in thecell culture supernatant determined by ELISA.

[0228] For intracytoplasmic staining for cytokines, monocytes arecultured (1 million/ml) in Yssel's medium in the absence or presence ofIL-B60 and LPS (E. coli 0127:B8 Difco) and 10 mg/ml Brefeldin A(Epicentre technologies Madison Wis.) for 12 hrs. Cells are washed inPBS and incubated in 2% formaldehyde/PBS solution for 20 minutes at RT.Subsequently cells are washed, resuspended in permeabilization buffer(0.5% saponin (Sigma) in PBS/BSA (0.5%)/Azide (1 mM)) and incubated for20 minutes at RT. Cells (2×10⁵) are centrifuged and resuspended in 20 mldirectly conjugated anti-cytokine mAbs diluted 1:10 in permeabilizationbuffer for 20 minutes at RT. The following antibodies can be used:IL-1α-PE (364-3B3-14); IL-6-PE (MQ2-13A5); TNFα-PE (MAb11); GM-CSF-PE(BVD2-21C11); and IL-12-PE (C11.5.14; Pharmingen San Diego, Calif.).Subsequently, cells are washed twice in permeabilization buffer and oncein PBS/BSA/Azide and analyzed on FACScan flow cytometer (BectonDickinson; Mountain View, Calif.).

[0229] E. Effects of IL-B60 on Proliferation of Human Peripheral BloodMononuclear Cells (PBMC)

[0230] Total PBMC are isolated from buffy coats of normal healthy donorsby centrifugation through ficoll-hypaque as described (Boyum, et al.).PBMC are cultured in 200 μl Yssel's medium (Gemini Bioproducts,Calabasas, Calif.) containing 1% human AB serum in 96 well plates(Falcon, Becton-Dickinson, N.J.) in the absence or presence of IL-B60.Cells are cultured in medium alone or in combination with 100 U/ml IL-2(R&D Systems) for 120 hours. 3H-Thymidine (0.1 mCi) is added during thelast six hours of culture and 3H-Thymidine incorporation determined byliquid scintillation counting.

[0231] The native, recombinant, and fusion proteins would be tested foragonist and antagonist activity in many other biological assay systems,e.g., on T-cells, B-cells, NK, macrophages, dendritic cells,hematopoietic progenitors, etc. Because of the IL-6 and G-CSF structuralrelationship, assays related to those activities should be analyzed

[0232] IL-B60 is evaluated for agonist or antagonist activity ontransfected cells expressing IL-6 or G-CSF receptor and controls. See,e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA 90, 11267-11271; Ho,et al. (1995) Mol. Cell. Biol. 15:5043-5053; and Liu, et al. (1994). J.Immunol. 152:1821-1829.

[0233] IL-B60 is evaluated for effect in macrophage/dendritic cellactivation and antigen presentation assays, T cell cytokine productionand proliferation in response to antigen or allogeneic stimulus. See,e.g., de Waal Malefyt et al. (1991) J. Exp. Med. 174:1209-1220; de WaalMalefyt et al. (199.1) J. Exp. Med. 174:915-924; Fiorentino, et al.(.1991) J. Immunol. 147, 3815-3822; Fiorentino, et al. (1991) J.Immunol. 146:3444-3451; and Groux, et al. (1996) J. Exp. Med. 184:19-29.

[0234] IL-B60 will also be evaluated for effects on NK cell stimulation.Assays may be based, e.g., on Hsu, et al. (1992) Internat. Immunol.4:563-569; and Schwarz, et al. (1994) J. Immunother. 16:95-104.

[0235] B cell growth and differentiation effects will be analyzed, e.g.,by the methodology described, e.g., in Defrance, et al. (1992). J. Exp.Med. 175:671-682; Rousset, et al. (1992) Proc. Nat'l Acad. Sci. USA89:1890-1893; including IgG2 and IgA2 switch factor assays. Note that,unlike COS7 supernatants, NIH3T3 and COP supernatants apparently do notinterfere with human B cell assays.

[0236] F. IL-B60 and CLF-1 Induce a Switch in NeurotransmitterProperties

[0237] Cholineric sympathetic neurons innervate at least three differenttargets: sweat glands, vasculature in skeletal muscle, and periosteum.Mature innervation of sympathetic neurons begins at the end of the firstpostnatal week and is characterized by the appearance of cholinericproperties. Cultures of sympathetic neurons were analyzed for theinduction of different neuromodulators, which specify the cholinergicphenotype. Cholecystokinin (CCK), vasoactive intestinal polypeptide(VIP), substance P (SP) and somatostatin (SOM) are upregulated afterstimulation of neurons with conditioned medium from IL-B60/CLF-1cotransfected cells or the CNTFR-IL-B60 fusion protein. Thus, thecomplex exhibits significant developmental biology function, and may beeffective in inducing certain aspects of neural development.

[0238] XI. Generation and Analysis of Genetically Altered Animals

[0239] Transgenic mice can be generated by standard methods. Suchanimals are useful to determine the effects of overexpression of thegene, in specific tissues, or completely throughout the organism. Suchmay provide interesting insight into development of the animal orparticular tissues in various stages. Moreover, the effect on variousresponses to biological stress can be evaluated. See, e.g., Hogan, etal. (1995) Manipulating the Mouse Embryo: A Laboratory Manual (2d ed.)Cold Spring Harbor Laboratory Press.

[0240] Adenovirus techniques are available for expression of the gene invarious cells and organs. See, e.g., Hitt, et al. (1997) Adv. Pharmacol.40:137-195; and literature from Quantum Biotechnologies, Montreal,Canada. Animals may be useful to determine the effects of the gene onvarious developmental or physiologically functional animal systems.

[0241] The genomic structure for the mouse IL-B60 has been determined. Astrategy for the production of IL-B60 knock-out mice can be developed,and appropriate constructs made.

[0242] All references cited herein are incorporated herein by referenceto the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety for all purposes.

[0243] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1 13 1 1790 DNA primate CDS (162)..(806) mat_peptide (213)..(806) 1ccgagcgaaa aaaacctgcg agtgggcctg gcggatggga ttattaaagc ttcgccggag 60ccgcggctcg ccctcccact ccgccagcct ccgggagagg agccgcaccc ggccggcccg 120gccccagccc catggacctc cgagcagggg actcgtgggg g atg tta gcg tgc ctg 176Met Leu Ala Cys Leu -15 tgc acg gtg ctc tgg cac ctc cct gca gtg cca gctctc aat cgc aca 224 Cys Thr Val Leu Trp His Leu Pro Ala Val Pro Ala LeuAsn Arg Thr -10 -5 -1 1 ggg gac cca ggg cct ggc ccc tcc atc cag aaa acctat gac ctc acc 272 Gly Asp Pro Gly Pro Gly Pro Ser Ile Gln Lys Thr TyrAsp Leu Thr 5 10 15 20 cgc tac ctg gag cac caa ctc cgc agc ttg gct gggacc tat ctg aac 320 Arg Tyr Leu Glu His Gln Leu Arg Ser Leu Ala Gly ThrTyr Leu Asn 25 30 35 tac ctg ggc ccc cct ttc aac gag cca gac ttc aac cctccc cgc ctg 368 Tyr Leu Gly Pro Pro Phe Asn Glu Pro Asp Phe Asn Pro ProArg Leu 40 45 50 ggg gca gag act ctg ccc agg gcc act gtt gac ttg gag gtgtgg cga 416 Gly Ala Glu Thr Leu Pro Arg Ala Thr Val Asp Leu Glu Val TrpArg 55 60 65 agc ctc aat gac aaa ctg cgg ctg acc cag aac tac gag gcc tacagc 464 Ser Leu Asn Asp Lys Leu Arg Leu Thr Gln Asn Tyr Glu Ala Tyr Ser70 75 80 cac ctt ctg tgt tac ttg cgt ggc ctc aac cgt cag gct gcc act gct512 His Leu Leu Cys Tyr Leu Arg Gly Leu Asn Arg Gln Ala Ala Thr Ala 8590 95 100 gag ctg cgc cgc agc ctg gcc cac ttc tgc acc agc ctc cag ggcctg 560 Glu Leu Arg Arg Ser Leu Ala His Phe Cys Thr Ser Leu Gln Gly Leu105 110 115 ctg ggc agc att gcg ggc gtc atg gca gct ctg ggc tac cca ctgccc 608 Leu Gly Ser Ile Ala Gly Val Met Ala Ala Leu Gly Tyr Pro Leu Pro120 125 130 cag ccg ctg cct ggg act gaa ccc act tgg act cct ggc cct gcccac 656 Gln Pro Leu Pro Gly Thr Glu Pro Thr Trp Thr Pro Gly Pro Ala His135 140 145 agt gac ttc ctc cag aag atg gac gac ttc tgg ctg ctg aag gagctg 704 Ser Asp Phe Leu Gln Lys Met Asp Asp Phe Trp Leu Leu Lys Glu Leu150 155 160 cag acc tgg ctg tgg cgc tcg gcc aag gac ttc aac cgg ctc aagaag 752 Gln Thr Trp Leu Trp Arg Ser Ala Lys Asp Phe Asn Arg Leu Lys Lys165 170 175 180 aag atg cag cct cca gca gct gca gtc acc ctg cac ctg ggggct cat 800 Lys Met Gln Pro Pro Ala Ala Ala Val Thr Leu His Leu Gly AlaHis 185 190 195 ggc ttc tgacttctga ccttctcctc ttcgctcccc cttcaaaccctgctcccact 856 Gly Phe ttgtgagagc cagccctgta tgccaacacc tgttgagccaggagacagaa gctgtgagcc 916 tctggccctt tcctggaccg gctgggcgtg tgatgcgatcagccctgtct cctccccacc 976 tcccaaaggt ctaccgagct ggggaggagg tacagtaggccctgtcctgt cctgtttcta 1036 caggaagtca tgctcgaggg agtgtgaagt ggttcaggttggtgcagagg cgctcatggc 1096 ctcctgcttc ttgcctacca cttggccagt gcccacccagcccctcaggt ggcacatctg 1156 gagggcaggg gttgaggggc caccaccaca catgcctttctggggtgaag ccctttggct 1216 gccccactct ccttggatgg gtgttgctcc cttatccccaaatcactcta tacatccaat 1276 tcaggaaaca aacatggtgg caattctaca caaaaagagatgagattaac agtgcagggt 1336 tggggtctgc attggaggtg ccctataaac cagaagagaaaatactgaaa gcacaggggc 1396 agggacagac cagaccagac ccaggagtct ccaaagcacagagtggcaaa caaaacccga 1456 gctgagcatc aggaccttgc ctcgaattgt cttccagtattacggtgcct cttctctgcc 1516 ccctttccca gggtatctgt gggttgccag gctggggagggcaaccatag ccacaccaca 1576 ggatttcctg aaagtttaca atgcagtagc attttggggtgtagggtggc agctccccaa 1636 ggccctgccc cccagcccca cccactcatg actctaagtgtgttgtatta atatttattt 1696 atttggagat gttatttatt agatgatatt tattgcagaatttctattct tgtattaaca 1756 aataaaatgc ttgccccaga acaaaaaaaa aaaa 1790 2215 PRT primate 2 Met Leu Ala Cys Leu Cys Thr Val Leu Trp His Leu ProAla Val Pro -15 -10 -5 Ala Leu Asn Arg Thr Gly Asp Pro Gly Pro Gly ProSer Ile Gln Lys -1 1 5 10 15 Thr Tyr Asp Leu Thr Arg Tyr Leu Glu His GlnLeu Arg Ser Leu Ala 20 25 30 Gly Thr Tyr Leu Asn Tyr Leu Gly Pro Pro PheAsn Glu Pro Asp Phe 35 40 45 Asn Pro Pro Arg Leu Gly Ala Glu Thr Leu ProArg Ala Thr Val Asp 50 55 60 Leu Glu Val Trp Arg Ser Leu Asn Asp Lys LeuArg Leu Thr Gln Asn 65 70 75 Tyr Glu Ala Tyr Ser His Leu Leu Cys Tyr LeuArg Gly Leu Asn Arg 80 85 90 95 Gln Ala Ala Thr Ala Glu Leu Arg Arg SerLeu Ala His Phe Cys Thr 100 105 110 Ser Leu Gln Gly Leu Leu Gly Ser IleAla Gly Val Met Ala Ala Leu 115 120 125 Gly Tyr Pro Leu Pro Gln Pro LeuPro Gly Thr Glu Pro Thr Trp Thr 130 135 140 Pro Gly Pro Ala His Ser AspPhe Leu Gln Lys Met Asp Asp Phe Trp 145 150 155 Leu Leu Lys Glu Leu GlnThr Trp Leu Trp Arg Ser Ala Lys Asp Phe 160 165 170 175 Asn Arg Leu LysLys Lys Met Gln Pro Pro Ala Ala Ala Val Thr Leu 180 185 190 His Leu GlyAla His Gly Phe 195 3 648 DNA primate CDS (1)..(645) mat_peptide(52)..(645) 3 atg tta gct tgc cta tgc acg gtg ctg tgg cac ctc cct gcagtg cca 48 Met Leu Ala Cys Leu Cys Thr Val Leu Trp His Leu Pro Ala ValPro -15 -10 -5 gct ctt aat cgc aca gga gat cca ggc cct ggc ccc tcc atccag aaa 96 Ala Leu Asn Arg Thr Gly Asp Pro Gly Pro Gly Pro Ser Ile GlnLys -1 1 5 10 15 acc tat gac ctc acc cgc tac ctg gag cat caa ctc cgc agctta gct 144 Thr Tyr Asp Leu Thr Arg Tyr Leu Glu His Gln Leu Arg Ser LeuAla 20 25 30 ggg acc tac ctg aac tac ctg ggg ccc cct ttc aac gag cct gacttc 192 Gly Thr Tyr Leu Asn Tyr Leu Gly Pro Pro Phe Asn Glu Pro Asp Phe35 40 45 aat cct cct cga ctg ggg gca gaa act ctg ccc agg gcc acg gtc aac240 Asn Pro Pro Arg Leu Gly Ala Glu Thr Leu Pro Arg Ala Thr Val Asn 5055 60 ttg gaa gtg tgg cga agc ctc aat gac agg ctg cgg ctg acc cag aac288 Leu Glu Val Trp Arg Ser Leu Asn Asp Arg Leu Arg Leu Thr Gln Asn 6570 75 tat gag gcg tac agt cac ctc ctg tgt tac ttg cgt ggc ctc aac cgt336 Tyr Glu Ala Tyr Ser His Leu Leu Cys Tyr Leu Arg Gly Leu Asn Arg 8085 90 95 cag gct gcc aca gct gaa ctc cga cgt agc ctg gcc cac ttc tgt acc384 Gln Ala Ala Thr Ala Glu Leu Arg Arg Ser Leu Ala His Phe Cys Thr 100105 110 agc ctc cag ggc ctg ctg ggc agc att gca ggt gtc atg gcg acg ctt432 Ser Leu Gln Gly Leu Leu Gly Ser Ile Ala Gly Val Met Ala Thr Leu 115120 125 ggc tac cca ctg ccc cag cct ctg cca ggg act gag cca gcc tgg gcc480 Gly Tyr Pro Leu Pro Gln Pro Leu Pro Gly Thr Glu Pro Ala Trp Ala 130135 140 cct ggc cct gcc cac agt gac ttc ctc cag aag atg gat gac ttc tgg528 Pro Gly Pro Ala His Ser Asp Phe Leu Gln Lys Met Asp Asp Phe Trp 145150 155 ctg ctg aag gag ctg cag acc tgg cta tgg cgt tca gcc aag gac ttc576 Leu Leu Lys Glu Leu Gln Thr Trp Leu Trp Arg Ser Ala Lys Asp Phe 160165 170 175 aac cgg ctt aag aag aag atg cag cct cca gca gct tca gtc accctg 624 Asn Arg Leu Lys Lys Lys Met Gln Pro Pro Ala Ala Ser Val Thr Leu180 185 190 cac ttg gag gcc cat ggt ttc tga 648 His Leu Glu Ala His GlyPhe 195 4 215 PRT primate 4 Met Leu Ala Cys Leu Cys Thr Val Leu Trp HisLeu Pro Ala Val Pro -15 -10 -5 Ala Leu Asn Arg Thr Gly Asp Pro Gly ProGly Pro Ser Ile Gln Lys -1 1 5 10 15 Thr Tyr Asp Leu Thr Arg Tyr Leu GluHis Gln Leu Arg Ser Leu Ala 20 25 30 Gly Thr Tyr Leu Asn Tyr Leu Gly ProPro Phe Asn Glu Pro Asp Phe 35 40 45 Asn Pro Pro Arg Leu Gly Ala Glu ThrLeu Pro Arg Ala Thr Val Asn 50 55 60 Leu Glu Val Trp Arg Ser Leu Asn AspArg Leu Arg Leu Thr Gln Asn 65 70 75 Tyr Glu Ala Tyr Ser His Leu Leu CysTyr Leu Arg Gly Leu Asn Arg 80 85 90 95 Gln Ala Ala Thr Ala Glu Leu ArgArg Ser Leu Ala His Phe Cys Thr 100 105 110 Ser Leu Gln Gly Leu Leu GlySer Ile Ala Gly Val Met Ala Thr Leu 115 120 125 Gly Tyr Pro Leu Pro GlnPro Leu Pro Gly Thr Glu Pro Ala Trp Ala 130 135 140 Pro Gly Pro Ala HisSer Asp Phe Leu Gln Lys Met Asp Asp Phe Trp 145 150 155 Leu Leu Lys GluLeu Gln Thr Trp Leu Trp Arg Ser Ala Lys Asp Phe 160 165 170 175 Asn ArgLeu Lys Lys Lys Met Gln Pro Pro Ala Ala Ser Val Thr Leu 180 185 190 HisLeu Glu Ala His Gly Phe 195 5 203 PRT rodent 5 Met Lys Val Leu Ala AlaGly Ile Val Pro Leu Leu Leu Leu Val Leu 1 5 10 15 His Trp Lys His GlyAla Gly Ser Pro Leu Pro Ile Thr Pro Val Asn 20 25 30 Ala Thr Cys Ala IleArg His Pro Cys His Gly Asn Leu Met Asn Gln 35 40 45 Ile Lys Asn Gln LeuAla Gln Leu Asn Gly Ser Ala Asn Ala Leu Phe 50 55 60 Ile Ser Tyr Tyr ThrAla Gln Gly Glu Pro Phe Pro Asn Asn Val Glu 65 70 75 80 Lys Leu Cys AlaPro Asn Met Thr Asp Phe Pro Ser Phe His Gly Asn 85 90 95 Gly Thr Glu LysThr Lys Leu Val Glu Leu Tyr Arg Met Val Ala Tyr 100 105 110 Leu Ser AlaSer Leu Thr Asn Ile Thr Arg Asp Gln Lys Val Leu Asn 115 120 125 Pro ThrAla Val Ser Leu Gln Val Lys Leu Asn Ala Thr Ile Asp Val 130 135 140 MetArg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Asn Lys Tyr 145 150 155160 Arg Val Gly His Val Asp Val Pro Pro Val Pro Asp His Ser Asp Lys 165170 175 Glu Ala Phe Gln Arg Lys Lys Leu Gly Cys Gln Leu Leu Gly Thr Tyr180 185 190 Lys Gln Val Ile Ser Val Val Val Gln Ala Phe 195 200 6 202PRT primate 6 Met Lys Val Leu Ala Ala Gly Val Val Pro Leu Leu Leu ValLeu His 1 5 10 15 Trp Lys His Gly Ala Gly Ser Pro Leu Pro Ile Thr ProVal Asn Ala 20 25 30 Thr Cys Ala Ile Arg His Pro Cys His Asn Asn Leu MetAsn Gln Ile 35 40 45 Arg Ser Gln Leu Ala Gln Leu Asn Gly Ser Ala Asn AlaLeu Phe Ile 50 55 60 Leu Tyr Tyr Thr Ala Gln Gly Glu Pro Phe Pro Asn AsnLeu Asp Lys 65 70 75 80 Leu Cys Gly Pro Asn Val Thr Asp Phe Pro Pro PheHis Ala Asn Gly 85 90 95 Thr Glu Lys Ala Lys Leu Val Glu Leu Tyr Arg IleVal Val Tyr Leu 100 105 110 Gly Thr Ser Leu Gly Asn Ile Thr Arg Asp GlnLys Ile Leu Asn Pro 115 120 125 Ser Ala Leu Ser Leu His Ser Lys Leu AsnAla Thr Ala Asp Ile Leu 130 135 140 Arg Gly Leu Leu Ser Asn Val Leu CysArg Leu Cys Ser Lys Tyr His 145 150 155 160 Val Gly His Val Asp Val ThrTyr Gly Pro Asp Thr Ser Gly Lys Asp 165 170 175 Val Phe Gln Lys Lys LysLeu Gly Cys Gln Leu Leu Gly Lys Tyr Lys 180 185 190 Gln Ile Ile Ala ValLeu Ala Gln Ala Phe 195 200 7 201 PRT primate 7 Met Ser Arg Arg Glu GlySer Leu Glu Asp Pro Gln Thr Asp Ser Ser 1 5 10 15 Val Ser Leu Leu ProHis Leu Glu Ala Lys Ile Arg Gln Thr His Ser 20 25 30 Leu Ala His Leu LeuThr Lys Tyr Ala Glu Gln Leu Leu Gln Glu Tyr 35 40 45 Val Gln Leu Gln GlyAsp Pro Phe Gly Leu Pro Ser Phe Ser Pro Pro 50 55 60 Arg Leu Pro Val AlaGly Leu Ser Ala Pro Ala Pro Ser His Ala Gly 65 70 75 80 Leu Pro Val HisGlu Arg Leu Arg Leu Asp Ala Ala Ala Leu Ala Ala 85 90 95 Leu Pro Pro LeuLeu Asp Ala Val Cys Arg Arg Gln Ala Glu Leu Asn 100 105 110 Pro Arg AlaPro Arg Leu Leu Arg Arg Leu Glu Asp Ala Ala Arg Gln 115 120 125 Ala ArgAla Leu Gly Ala Ala Val Glu Ala Leu Leu Ala Ala Leu Gly 130 135 140 AlaAla Asn Arg Gly Pro Arg Ala Glu Pro Pro Ala Ala Thr Ala Ser 145 150 155160 Ala Ala Ser Ala Thr Gly Val Phe Pro Ala Lys Val Leu Gly Leu Arg 165170 175 Val Cys Gly Leu Tyr Arg Glu Trp Leu Ser Arg Thr Glu Gly Asp Leu180 185 190 Gly Gln Leu Leu Pro Gly Gly Ser Ala 195 200 8 203 PRT rodent8 Met Ser Gln Arg Glu Gly Ser Leu Glu Asp His Gln Thr Asp Ser Ser 1 5 1015 Ile Ser Phe Leu Pro His Leu Glu Ala Lys Ile Arg Gln Thr His Asn 20 2530 Leu Ala Arg Leu Leu Thr Lys Tyr Ala Glu Gln Leu Leu Glu Glu Tyr 35 4045 Val Gln Gln Gln Gly Glu Pro Phe Gly Leu Pro Gly Phe Ser Pro Pro 50 5560 Arg Leu Pro Leu Ala Gly Leu Ser Gly Pro Ala Pro Ser His Ala Gly 65 7075 80 Leu Pro Val Ser Glu Arg Leu Arg Gln Asp Ala Ala Ala Leu Ser Val 8590 95 Leu Pro Ala Leu Leu Asp Ala Val Arg Arg Arg Gln Ala Glu Leu Asn100 105 110 Pro Arg Ala Pro Arg Leu Leu Arg Ser Leu Glu Asp Ala Ala ArgGln 115 120 125 Val Arg Ala Leu Gly Ala Ala Val Glu Thr Val Leu Ala AlaLeu Gly 130 135 140 Ala Ala Ala Arg Gly Pro Gly Pro Glu Pro Val Thr ValAla Thr Leu 145 150 155 160 Phe Thr Ala Asn Ser Thr Ala Gly Ile Phe SerAla Lys Val Leu Gly 165 170 175 Phe His Val Cys Gly Leu Tyr Gly Glu TrpVal Ser Arg Thr Glu Gly 180 185 190 Asp Leu Gly Gln Leu Val Pro Gly GlyVal Ala 195 200 9 200 PRT primate 9 Met Ala Phe Thr Glu His Ser Pro LeuThr Pro His Arg Arg Asp Leu 1 5 10 15 Cys Ser Arg Ser Ile Trp Leu AlaArg Lys Ile Arg Ser Asp Leu Thr 20 25 30 Ala Leu Thr Glu Ser Tyr Val LysHis Gln Gly Leu Asn Lys Asn Ile 35 40 45 Asn Leu Asp Ser Ala Asp Gly MetPro Val Ala Ser Thr Asp Gln Trp 50 55 60 Ser Glu Leu Thr Glu Ala Glu ArgLeu Gln Glu Asn Leu Gln Ala Tyr 65 70 75 80 Arg Thr Phe His Val Leu LeuAla Arg Leu Leu Glu Asp Gln Gln Val 85 90 95 His Phe Thr Pro Thr Glu GlyAsp Phe His Gln Ala Ile His Thr Leu 100 105 110 Leu Leu Gln Val Ala AlaPhe Ala Tyr Gln Ile Glu Glu Leu Met Ile 115 120 125 Leu Leu Glu Tyr LysIle Pro Arg Asn Glu Ala Asp Gly Met Pro Ile 130 135 140 Asn Val Gly AspGly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys 145 150 155 160 Val LeuGln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu 165 170 175 ArgPhe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His 180 185 190Tyr Ile Ala Asn Asn Lys Lys Met 195 200 10 198 PRT rodent 10 Met Ala PheAla Glu Gln Ser Pro Leu Thr Leu His Arg Arg Asp Leu 1 5 10 15 Cys SerArg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr 20 25 30 Ala LeuMet Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile 35 40 45 Ser LeuAsp Ser Val Asp Gly Val Pro Val Ala Ser Thr Asp Arg Trp 50 55 60 Ser GluMet Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr 65 70 75 80 ArgThr Phe Gln Gly Met Leu Thr Lys Leu Leu Glu Asp Gln Arg Val 85 90 95 HisPhe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu 100 105 110Thr Leu Gln Val Ser Ala Phe Ala Tyr Gln Leu Glu Glu Leu Met Ala 115 120125 Leu Leu Glu Gln Lys Val Pro Glu Lys Glu Ala Asp Gly Met Pro Val 130135 140 Thr Ile Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys145 150 155 160 Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile HisAsp Leu 165 170 175 Arg Val Ile Ser Ser His His Met Gly Ile Ser Ala HisGlu Ser His 180 185 190 Tyr Gly Ala Lys Gln Met 195 11 208 PRT primate11 Met Thr His Leu Ser Leu Leu Gly Pro Leu Pro Cys Val Arg Thr Ser 1 510 15 Gln Gln Leu Pro Glu Thr Gln Gln Val Thr Thr Pro Gly Lys Lys Pro 2025 30 Val Ser Val Gly Arg Arg Glu Val Arg Val Pro Gly Thr Ala Leu Val 3540 45 Pro Ser Leu Leu Ser Val Ser Val Leu Leu Gln Leu Gln Tyr Gln Gly 5055 60 Ser Pro Phe Ser Asp Pro Gly Phe Ser Ala Pro Glu Leu Gln Leu Ser 6570 75 80 Ser Leu Pro Pro Ala Thr Ala Phe Phe Lys Thr Trp His Ala Leu Asp85 90 95 Asp Gly Glu Arg Leu Ser Leu Ala Gln Arg Ala Ile Asp Pro His Leu100 105 110 Gln Leu Val Glu Asp Asp Gln Ser Asp Leu Asn Pro Gly Ser ProIle 115 120 125 Leu Pro Ala Gln Leu Gly Ala Ala Arg Leu Arg Ala Gln GlyPro Leu 130 135 140 Gly Asn Met Ala Ala Ile Met Thr Ala Leu Gly Leu ProIle Pro Pro 145 150 155 160 Glu Glu Asp Thr Pro Gly Leu Ala Ala Phe GlyAla Ser Ala Phe Glu 165 170 175 Arg Lys Cys Arg Gly Tyr Val Val Thr ArgGlu Tyr Gly His Trp Thr 180 185 190 Asp Arg Ala Val Arg Asp Leu Ala LeuLeu Lys Ala Lys Tyr Ser Ala 195 200 205 12 410 PRT primate 12 Met ProAla Gly Arg Arg Gly Pro Ala Ala Gln Ser Ala Arg Arg Pro 1 5 10 15 ProPro Leu Leu Pro Leu Leu Leu Leu Leu Cys Val Leu Gly Ala Pro 20 25 30 ArgAla Gly Ser Gly Ala His Thr Ala Val Ile Ser Pro Gln Asp Pro 35 40 45 ThrLeu Leu Ile Gly Ser Ser Leu Leu Ala Thr Cys Ser Val His Gly 50 55 60 AspPro Pro Gly Ala Thr Ala Glu Gly Leu Tyr Trp Thr Leu Asn Gly 65 70 75 80Arg Arg Leu Pro Pro Glu Leu Ser Arg Val Leu Asn Ala Ser Thr Leu 85 90 95Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gln Arg Ser Gly Asp 100 105110 Asn Leu Val Cys His Ala Arg Asp Gly Ser Ile Leu Ala Gly Ser Cys 115120 125 Leu Tyr Val Gly Leu Pro Pro Glu Lys Pro Val Asn Ile Ser Cys Trp130 135 140 Ser Lys Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly AlaHis 145 150 155 160 Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys TyrLys Leu Arg 165 170 175 Trp Tyr Gly Gln Asp Asn Thr Cys Glu Glu Tyr HisThr Val Gly Pro 180 185 190 His Ser Cys His Ile Pro Lys Asp Leu Ala LeuPhe Thr Pro Tyr Glu 195 200 205 Ile Trp Val Glu Ala Thr Asn Arg Leu GlySer Ala Arg Ser Asp Val 210 215 220 Leu Thr Leu Asp Ile Leu Asp Val ValThr Thr Asp Pro Pro Pro Asp 225 230 235 240 Val His Val Ser Arg Val GlyGly Leu Glu Asp Gln Leu Ser Val Arg 245 250 255 Trp Val Ser Pro Pro AlaLeu Lys Asp Phe Leu Phe Gln Ala Lys Tyr 260 265 270 Gln Ile Arg Tyr ArgVal Glu Asp Ser Val Asp Trp Lys Val Val Asp 275 280 285 Asp Val Ser AsnGln Thr Ser Cys Arg Leu Ala Gly Leu Lys Pro Gly 290 295 300 Thr Val TyrPhe Val Gln Val Arg Cys Asn Pro Phe Gly Ile Tyr Gly 305 310 315 320 SerLys Lys Ala Gly Ile Trp Ser Glu Trp Ser His Pro Thr Ala Ala 325 330 335Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro Gly Gly Gly Ala Cys Glu 340 345350 Pro Arg Gly Gly Glu Pro Ser Ser Gly Pro Val Arg Arg Glu Leu Lys 355360 365 Gln Phe Leu Gly Trp Leu Lys Lys His Ala Tyr Cys Ser Asn Leu Ser370 375 380 Phe Arg Leu Tyr Asp Gln Trp Arg Ala Trp Met Gln Lys Ser HisLys 385 390 395 400 Thr Arg Asn Gln Val Leu Pro Asp Lys Leu 405 410 13407 PRT rodent 13 Arg Pro Leu Ser Ser Leu Trp Ser Pro Leu Leu Leu CysVal Leu Gly 1 5 10 15 Val Pro Arg Gly Gly Ser Gly Ala His Thr Ala ValIle Ser Pro Gln 20 25 30 Asp Pro Thr Leu Leu Ile Gly Ser Ser Leu Gln AlaThr Cys Ser Ile 35 40 45 His Gly Asp Thr Pro Gly Ala Thr Ala Glu Gly LeuTyr Trp Thr Leu 50 55 60 Asn Gly Arg Arg Leu Pro Ser Leu Ser Arg Leu LeuAsn Thr Ser Thr 65 70 75 80 Leu Ala Leu Ala Leu Ala Asn Leu Asn Gly SerArg Gln Gln Ser Gly 85 90 95 Asp Asn Leu Val Cys His Ala Arg Asp Gly SerIle Leu Ala Gly Ser 100 105 110 Cys Leu Tyr Val Gly Leu Pro Pro Glu LysPro Phe Asn Ile Ser Cys 115 120 125 Trp Ser Arg Asn Met Lys Asp Leu ThrCys Arg Trp Thr Pro Gly Ala 130 135 140 His Gly Glu Thr Phe Leu His ThrAsn Tyr Ser Leu Lys Tyr Lys Leu 145 150 155 160 Arg Trp Tyr Gly Gln AspAsn Thr Cys Glu Glu Tyr His Thr Val Gly 165 170 175 Pro His Ser Cys HisIle Pro Lys Asp Leu Ala Leu Phe Thr Pro Tyr 180 185 190 Glu Ile Trp ValGlu Ala Thr Asn Arg Leu Gly Ser Ala Arg Ser Asp 195 200 205 Val Leu ThrLeu Asp Val Leu Asp Val Val Thr Thr Asp Pro Pro Pro 210 215 220 Asp ValHis Val Ser Arg Val Gly Gly Leu Glu Asp Gln Leu Ser Val 225 230 235 240Arg Trp Val Ser Pro Pro Ala Leu Lys Asp Phe Leu Phe Gln Ala Lys 245 250255 Tyr Gln Ile Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys Val Val 260265 270 Asp Asp Val Ser Asn Gln Thr Ser Cys Arg Leu Ala Gly Leu Lys Pro275 280 285 Gly Thr Val Tyr Phe Val Gln Val Arg Cys Asn Pro Phe Gly IleTyr 290 295 300 Gly Ser Lys Lys Ala Gly Ile Trp Ser Glu Trp Ser His ProThr Ala 305 310 315 320 Ala Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro GlyGly Gly Val Cys 325 330 335 Glu Pro Arg Gly Gly Glu Pro Ser Ser Gly ProVal Arg Arg Glu Leu 340 345 350 Lys Gln Phe Leu Gly Trp Leu Lys Lys HisAla Tyr Cys Ser Asn Leu 355 360 365 Ser Phe Arg Leu Tyr Asp Gln Trp ArgAla Trp Met Gln Lys Ser His 370 375 380 Lys Thr Arg Asn Gln Asp Glu GlyIle Leu Pro Ser Gly Arg Arg Gly 385 390 395 400 Ala Ala Arg Gly Pro AlaGly 405

What is claimed is:
 1. An isolated soluble complex comprising at least 6amino acids of the mature protein portion of SEQ ID NO: 2 or 4, and: a)at least 6 amino acids of the mature protein portion of SEQ ID NO: 12 or13; or b) at least 6 amino acids of the mature protein portion of theCNTF-R.
 2. The complex of claim 1, wherein said complex: a) comprises arecombinant polypeptide of mature SEQ ID NO: 2 or 4; b) comprises arecombinant polypeptide of mature SEQ ID NO: 12 or 13; c) comprises arecombinant polypeptide of mature CNTF-R; d) comprises both arecombinant polypeptide of mature SEQ ID NO: 2 or 4, and a recombinantpolypeptide of mature SEQ ID NO: 12 or 13; e) comprises both arecombinant polypeptide of mature SEQ ID NO: 2 or 4, and a recombinantpolypeptide of mature CNTF-R; f) is detectably labeled; g) is in abuffered solution; or h) is in a sterile solution.
 3. The complex ofclaim 1, which: a) comprises a mature IL-B60 polypeptide; b) comprises amature CLF-1 polypeptide; c) comprises a mature CNTF-R polypeptide; d)exhibits at least four nonoverlapping segments of at least seven aminoacids of SEQ ID NO: 2 or 4; e) exhibits epitopes from both primate L-B60and primate CLF-1; f) exhibits epitopes from both primate L-B60 andprimate CNTF-R; g) is not glycosylated; h) is attached to a solidsubstrate; i) is conjugated to another chemical moiety; or j) comprisesa detection or purification tag, including a FLAG, His6, or Ig sequence.4. A kit comprising said complex of claim 1, and: a) a compartmentcomprising said complex; or b) instructions for use or disposal ofreagents in said kit.
 5. An isolated or recombinant polypeptidecomprising: a) a first segment comprising at least seven amino acidsidentical to segments of SEQ ID NO: 2 or 4, and a second segmentcomprising at least seven amino acids identical to segments of matureSEQ ID NO: 12 or 13; b) at least two distinct nonoverlapping segments ofat least five amino acids identical to segments of mature SEQ ID NO: 2or 4, and a third segment comprising at least seven amino acidsidentical to segments of mature SEQ ID NO: 12 or 13; c) at least onesegment comprising at least seven amino acids identical to segments ofmature SEQ ID NO: 2 or 4, and two distinct nonoverlapping segments of atleast five amino acids identical to segments of mature SEQ ID NO: 12 or13; d) a first segment comprising at least seven amino acids identicalto segments of SEQ ID NO: 2 or 4, and a second segment comprising atleast seven amino acids identical to segments of mature primate CNTF-R;e) at least two distinct nonoverlapping segments of at least five aminoacids identical to segments of mature SEQ ID NO: 2 or 4, and a thirdsegment comprising at least seven amino acids identical to segments ofmature primate CNTF-R; or f) at least one segment comprising at leastseven amino acids identical to segments of mature SEQ ID NO: 2 or 4, andtwo distinct nonoverlapping segments of at least five amino acidsidentical to segments of mature primate CNTF-R.
 6. The polypeptide ofclaim 5, wherein said distinct nonoverlapping segments of identity: a)include one of at least eight amino acids; b) include one of at leastfive amino acids and a second of at least six amino acids; c) include atleast three segments of at least four, five, and six amino acids, or d)include one of at least twelve amino acids.
 7. The polypeptide of claim5, which: a) comprises a mature IL-B60 sequence; b) comprises a matureCLF-1 sequence; c) comprises a mature CNTF-R sequence; d) exhibits atleast four nonoverlapping segments of at least seven amino acids of SEQID NO: 2 or 4; e) has a length at least about 30 amino acids; f)exhibits epitopes from both primate IL-B60 and primate CLF-1; g)exhibits epitopes from both primate IL-B60 and primate CNTF-R; h) is notglycosylated; i) has a molecular weight of at least 30 kD; j) is asynthetic polypeptide; k) is attached to a solid substrate; l) isconjugated to another chemical moiety; or m) comprises a detection orpurification tag, including a FLAG, His6, or Ig sequence.
 8. Acomposition comprising: a) substantially pure combination of IL-B60 andCLF-1; b) substantially pure combination of IL-B60 and CNTF-R; c) asterile polypeptide of claim 5; or d) said polypeptide of claim 5 and acarrier, wherein said carrier is: i) an aqueous compound, includingwater, saline, and/or buffer; and/or ii) formulated for oral, rectal,nasal, topical, or parenteral administration.
 9. A kit comprising apolypeptide of claim 5, and: a) a compartment comprising saidpolypeptide; or b) instructions for use or disposal of reagents in saidkit.
 10. A method: a) of making an antibody which recognizes a complexof claim 1, comprising inducing an immune response in an animal withsaid complex; b) of immunoselecting antibodies, comprising contacting apopulation of antibodies to a complex of claim 1, and separatingantibodies that bind from those which do not bind; or c) of formulatinga composition, comprising admixing a complex of claim 1 with a carrier.11. A binding compound comprising an antigen binding site from anantibody, which antibody specifically binds said complex of claim 2d or2e, but not to any of said mature polypeptides of SEQ ID NO: 2, 4, 12,13, or CNTF-R.
 12. The binding compound of claim 11, wherein: a) saidbinding compound is: i) in a container; ii) an Fv, Fab, or Fab2fragment; or iii) conjugated to another chemical moiety; or b) saidantibody: i) is raised against a substantially pure complex of IL-B60with CLF-1; ii) is raised against a substantially pure complex of IL-B60with CNTF-R; iii) is immunoselected; iv) is a polyclonal antibody; v)exhibits a Kd to antigen of at least 30 μM; vi) is attached to a solidsubstrate, including a bead or plastic membrane; vii) is in a sterilecomposition; or viii) is detectably labeled, including a radioactive orfluorescent label.
 13. A composition comprising: a) a sterile bindingcompound of claim 12, or b) said binding compound of claim 12 and acarrier, wherein said carrier is: i) an aqueous compound, includingwater, saline, and/or buffer; and/or ii) formulated for oral, rectal,nasal, topical, or parenteral administration.
 14. A kit comprising saidbinding compound of claim 11, and: a) a compartment comprising saidbinding compound; or b) instructions for use or disposal of reagents insaid kit.
 15. A method of producing an antigen:antibody complex,comprising contacting under appropriate conditions a primate complexcomprising: a) IL-B60 and CLF-1 polypeptides; or b) IL-B60 and CNTF-Rpolypeptides; with an antibody of claim 11, thereby allowing saidcomplex to form.
 16. The method of claim 15, wherein: a) said complex ispurified from other cytokines; b) said complex is purified from otherantibody; c) said contacting is with a sample comprising a cytokine; d)said contacting allows quantitative detection of said antigen; e) saidcontacting is with a sample comprising said antibody; or f) saidcontacting allows quantitative detection of said antibody.
 17. Anisolated or recombinant nucleic acid: a) encoding said amino acidportions of claim 5; b) encoding said amino acid portions of claim 5,and comprise a segment at least 30 contiguous nucleotides from SEQ IDNO: 1 or 3; c) which will coexpress a segment of at least sevencontiguous amino acids from SEQ ID NO: 2 or 4, and a segment of at leastseven contiguous amino acids from SEQ ID NO: 12 or 13; or d) which willcoexpress a segment of at least seven contiguous amino acids from SEQ IDNO: 2 or 4, and a segment of at least seven contiguous amino acids fromCNTF-R.
 18. The nucleic acid of claim 17, which: a) encodes IL-B60 froma human; b) encodes CLF-1 from a human; c) encodes CNTF-R from a human;d) is an expression vector; e) further comprises an origin ofreplication; f) comprises a detectable label; g) comprises syntheticnucleotide sequence; or h) is less than 6 kb, preferably less than 3 kb.19. A cell comprising said recombinant nucleic acid of claim
 18. 20. Thecell of claim 19, wherein said cell is: a) a prokaryotic cell; b) aeukaryotic cell; c) a bacterial cell; d) a yeast cell; e) an insectcell; f) a mammalian cell; g) a mouse cell; h) a primate cell; or i) ahuman cell.
 21. A kit comprising said nucleic acid of claim 18, and: a)a compartment comprising said nucleic acid; b) a compartment furthercomprising a primate IL-B60 polypeptide; c) a compartment furthercomprising a primate CLF-1 polypeptide; d) a compartment furthercomprising a primate CNTF-R polypeptide; or e) instructions for use ordisposal of reagents in said kit.
 22. A method: a) of making a duplexnucleic acid, comprising contacting a nucleic acid of claim 17 with acomplementary nucleic acid under appropriate conditions, thereby formingsaid duplex; b) of expressing a polypeptide, comprising expressing saidnucleic acid of claim 17, thereby producing said polypeptide; or c) oftransfecting a cell, comprising contacting said cell under appropriateconditions with said nucleic acid of claim
 17. 23. An isolated orrecombinant nucleic acid which encodes at least 5 contiguous amino acidsof SEQ ID NO: 12, 13, or primate CNTF-R and: a) hybridizes under washconditions of 30 minutes at 30° C. and less than 2M salt to the codingportion of SEQ ID NO: 1; or b) exhibits identity over a stretch of atleast about 30 nucleotides to a primate IL-B60.
 24. The isolated nucleicacid of claim 23, wherein: a) said contiguous amino acids number atleast 8; b) said wash conditions are at 45° C. and/or 500 mM salt; or c)said stretch is at least 55 nucleotides.
 25. The recombinant nucleicacid of claim 23, wherein: a) said contiguous amino acids number atleast 12; b) said wash conditions are at 55° C. and/or 150 mM salt; orc) said stretch is at least 75 nucleotides.
 26. A method of modulatingphysiology or development of a cell or tissue culture cells comprisingcontacting said cell with an agonist or antagonist of a complexcomprising mammalian IL-B60 and: a) CLF-1; or b) CNTF-R.
 27. A methodof: a) producing a complex of claim 1, comprising coexpressing arecombinant IL-B60 with a recombinant CLF-1 or CNTF-R; b) increasing thesecretion of an IL-B60 polypeptide comprising expressing saidpolypeptide with CLF-1; or c) increasing the secretion of a CLF-1polypeptide, comprising expressing said CLF-1 with an IL-B60.
 28. Themethod of claim 27, wherein: a) said increasing is at least 3 fold; orb) said expressing is of a recombinant nucleic acid encoding one or bothof said polypeptide and CLF-1.
 29. A method of screening for a receptorwhich binds said complex of claim 1, comprising contacting said complexto a cell expressing said receptor under conditions allowing saidcomplex to bind to said receptor, thereby forming a detectableinteraction.
 30. The method of claim 29, wherein said interactionresults in a physiological response in said cell.